Methods for treating triple-negative breast cancer

ABSTRACT

The invention is directed to methods of treating TNBC in a patient by administering to the patient an agent that inhibits the expression or activity of cyclin-dependent kinase 19 (CDK19). In some embodiments, the agent may be a small molecule inhibitor, a polynucleotide (e.g., shRNA. siRNA), or a protein (e.g., an antibody). In some embodiments, the agent does not inhibit the activity or expression of CDK8.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit and is a continuation of Application No.16/648,088 filed Mar. 17, 2020, which is a national phase application ofPCT Application No. PCT/US2018/051489, filed Sep. 18, 2018, which claimsbenefit of U.S. Provisional Pat. Application No.: 62/560,140, filed Sep.18, 2017, which applications are incorporated by reference in theirentirety for all purposes.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under contractW81XWH-11-1-0287 awarded by the Department of Defense; under contractW81XWH-13-1-0281 awarded by the Department of Defense; and undercontract CA100225 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

REFERENCE TO A SEQUENCE LISTING

[0002A] The Sequence Listing written in file103182-1342710-000120US_Seq_Listing.xml created on Aug. 18, 2022, 81 KB,is hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

The invention relates to the field of biomedicine, e.g., oncology.

BACKGROUND

Triple-negative breast cancer (TNBC) is an aggressive breast cancersubtype disproportionately affecting younger women and associated withpoor prognoses. See Bauer et al. “Descriptive analysis of estrogenreceptor (ER)-negative, progesterone receptor (PR)-negative, andHER2-negative invasive breast cancer, the so-called triple-negativephenotype: a population-based study from the California cancer Registry”Cancer 109, 1721-1728, doi:10.1002/cncr.22618 (2007). Despite affecting20% of all breast cancer patients, there are currently no clinicallyapproved targeted therapies for these patients. There exists a need inthe art for effective methods of treating TNBC.

SUMMARY

The invention is directed to methods of treating TNBC in a patient byadministering to the patient an agent that inhibits the expression oractivity of cyclin-dependent kinase 19 (CDK19).

In one aspect, the invention features a method of treating a patientdiagnosed with triple-negative breast cancer (TNBC) by administering atherapeutically effective dose of an agent that inhibits expression oractivity of cyclin-dependent kinase 19 (CDK19) and achieves at least oneof a reduction in cachexia, increase in survival time, elongation intime to tumor progression, reduction in tumor mass, reduction in tumorburden and/or a prolongation in time to tumor metastasis, time to tumorrecurrence, tumor response, complete response, partial response, stabledisease, progressive disease, or progression free survival.

In another aspect, the invention features a method of treating a patientdiagnosed with triple-negative breast cancer (TNBC), wherein the canceris characterized by a tumor comprising EpCAM^(med/high)/CD10^(-/low)epithelial cells. The method includes administering a therapeuticallyeffective dose of an agent that inhibits cyclin-dependent kinase 19(CDK19) expression or activity, wherein the treatment reduces the numberof E_(P)CAM^(med/high)/ CD10^(-/low) cells in the tumor, reduces tonumber of E_(P)CAM ^(med/high) /CD10^(-/low) cells per unit volume ofthe tumor, or results in a reduction of the ratio ofEpCAMm^(ed/high)/CD1₀ ^(/low) epithelial cells to normal(EpCam^(Hi)/CD10⁻) epithelial cells in the tumor.

In yet another aspect, the invention features a method of reducingmetastasis of TNBC in a patient by administering a therapeuticallyeffective dose of an agent that inhibits expression or activity ofCDK19.

In some embodiments of all aspects of the invention described herein,the patient is treated with a combination therapy comprising (a) anagent that inhibits expression or activity of CDK19 and (b) radiationtherapy and/or chemotherapy.

In some embodiments, the method comprises detectingEpCAM^(med/hlgh)/CD10^(-/low) cells in a tissue sample from the patientprior to or after initiating therapy.

In some embodiments, the agent administered to the patient in themethods described herein does not significantly inhibit expression oractivity of CDK8. In some embodiments, the agent inhibits expression oractivity of CDK19 to a greater extent than it inhibits expression oractivity of CDK8.

In some embodiments of the methods describe herein, the agent is anucleic acid. In some embodiments, the agent is a protein. In someembodiments, the agent is a CRISPR/Cas9 system.

In some embodiments of the methods describe herein, the agent is a CDK19targeting shRNA.

In some embodiments of the methods describe herein, the agent is a CDK19targeting siRNA.

In some embodiments of the methods describe herein, the agent is a CDK19targeting shRNA or siRNA complementary or substantially complementary tothe 3′ UTR of CDK19, but not to the 3′UTR CDK8.

In some embodiments of the methods describe herein, the agent is a CDK19targeting shRNA or siRNA complementary or substantially complementary tothe coding region of CDK19, but not to the coding region of CDK8.

In some embodiments of the methods describe herein, the agent is a CDK19targeting shRNA or siRNA selected from: SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

In some embodiments, the agent binds CDK19 in the cytoplasm of a breastepithelial cell.

In another aspect, the invention also features a method of predictingthe likely therapeutic responsiveness of a subject with TNBC to a CDK19targeting agent. The method includes (a) quantitatingEpCAM^(med/high)/CD10^(-/low) cells in a tumor sample obtained from thesubject; (b) comparing the quantity of EpCAM^(med/high)/CD10^(-/low)cells in (a) to a reference value characteristic of tumors responsive toa CDK19 targeting therapy, and treating the patient with the CDK19targeting agent if the quantity of EpCAM^(med/high)/CD10^(-/low) cellsis equal to or exceeds the reference value. In some embodiments, theCDK19 targeting agent is an inhibitor of CDK19 expression or activity

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic for RNAi dropout viability screens. Two separatescreens were performed in a TNBC PDX (PDX-T1). Cells in one experimentwere grown in vitro as organoid colonies and in the other in vivo asPDXs in NSG mice.

FIGS. 1B-1D are graphs showing that CDK19 knockdown significantlydecreased the viability of TNBC cells (FIG. 1B: MDA-MB231 cells; FIG.1C: MDA-MB468 cells; and FIG. 1D: HS578T cells) assessed 4 days aftertransduction with control shRNA or CDK19 targeting shRNA (shCDK19-1,shCDK19-2).

FIG. 1E is a graph showing that CDK19 knockdown significantly decreasedthe formation of organoid colonies in PDX-T1.

FIG. 1F is a graph showing that CDK19 knockdown does not decrease theviability of non-transformed human mammary epithelial cells (HMEC).

FIGS. 1G-1J are graphs showing that CDK19 knockdown significantlyinhibits the proliferation of PDX tumors (FIG. 1G: PDX-T1; FIG. 1H:PDX-T2; FIG. 11 : PDX-T3; and FIG. 1J: PDX-T4) grown in NSG mice.

FIGS. 1K and 1L are bar graphs showing that CDK19 knockdown preventedtransduced (RFP positive) TNBC cells (FIG. 1K: PDX-T1 and FIG. 1LPDX-T4) from metastasizing to the lungs in mice.

FIG. 1M shows that in PDX tumors transduced with CDK19 shRNA (images inthe second and third rows), very little RFP (images in the last column)is visible. These tumors are composed primarily of un-transduced GFPpositive tumor cells (images in the middle column). PDX tumor cells werefirst labeled with green fluorescent protein (GFP) (middle column) andcells subsequently infected with either CDK19 shRNA or control shRNAwere additionally labeled with red fluorescent protein (RFP) (rightcolumn).

FIG. 1N shows representative images of mouse lungs with PDX-T1metastases. Lungs from mice with PDXs transduced with control shRNA (toprow), shCDK19-1 (middle row) or shCDK19-2 (bottom row) are shown. InPDX-T1, which normally metastasizes to the lung, CDK19 knockdowneliminated the detection of any lung metastases by those cells. Brightfield images (left column) show gross lung morphology, FITC images(middle column) identify metastatic tumor cells labeled with GFP, andmetastatic tumor cells subsequently infected with either CDK19 shRNA orcontrol shRNA were additionally labeled with red fluorescent protein(RFP) (right column).

FIG. 2A shows data from representative flow cytometry analyses of a TNBC(PDX-T1) using EpCAM and CD49f (left) or EpCAM and CD10 (right) as cellsurface markers.

FIG. 2B is a graph that compares the organoid colony formingcapabilities of the EpCAM^(med/high)/CD10^(-/low) andEPCAM^(low/med)/CD10^(low/+) cell sub-populations.

FIG. 2C is a table showing the number of tumors formed and the number ofinjections performed for six groups of PDX tumor cells. Populations andinjections where tumors formed are bolded. PDX tumor cells were isolatedby flow cytometry based on the expression of EpCAM and CD10 (as in FIG.2A, right)

FIGS. 2D-2G are bar graphs showing that CDK19 expression is higher inthe EpCAM^(med/high)/CD10^(-/low) cells compared to theEPCAM^(low/med)/CD10^(low/+) cells in PDX-T1, PDX-T2, and PDX-T8.

FIG. 3A includes Venn diagrams showing the number of genes upregulated(upper diagram) and downregulated (lower diagram) by CDK19 knockdown,CDK8 knockdown, or by both CDK19 and CDK8 (overlap region).

FIG. 3B is a Venn diagram of Hallmark gene sets enriched across thegenes upregulated (upper diagram) or downregulated (lower diagram) byCDK19 knockdown, CDK8 knockdown, or by both CDK19 and CDK8 knockdowns(overlap region) as determined by GSEA.

FIGS. 3C and 3D are graphs showing that CHIP-Seq signals across theCDK19KD-H3K27AcUP and CDK19KD-H3K27AcDOWN regions are significantlydifferent in the CDK19 knockdown samples compared to control.

FIGS. 3E and 3F are graphs showing a gene set enrichment analysis (GSEA)of CDK19KD-EnhancerUP and CDK19KD-EnhancerDOWN genes using averagedCDK19 knockdown versus control expression data.

FIG. 3G is a graph showing the hallmark gene sets identified as enrichedin Metascape analysis of the CDK19KD-EnhancerUP ‘core’ genes (top andmiddle bars) and CDK19KD-EnhancerDOWN ‘core’ (bottom bar) genes. Theindividual genes contributing to the enrichment of each hallmark geneset are shown to the right of each bar.

FIGS. 4A and 4B are graphs showing that in inducCDK19KD-PDX-Tl cells,induction of CDK19 shRNA by addition of doxycycline significantlydecreased the number of organoid colonies in the doxycycline treatmentgroup compared to control. Number of organoid colonies at Day 0 (FIG.4A) and Day 16 (FIG. 4B) after initiating doxycycline treatment isshown.

FIGS. 4C and 4D are graphs showing that the induction of CDK19 shRNA inpre-established tumors impaired tumor growth. The growth ofpre-established tumors in the doxycycline fed NSG mice and control NSGmice are shown for inducCDK19KD-PDX-Tl (FIG. 4C) and inducCDK19KD-PDX-T3(FIG. 4D).

FIG. 4E is a graph showing that CDK19 knockdown extends the survival ofNSG mice with PDX-T1 tumors.

FIG. 4F shows the chemical structure of CCT251921, an orallybioavailable selective inhibitor of CDK19 and CDK8.

FIG. 4G is a graph showing that the treatment of mice with CCT251921 bydaily oral gavage significantly impaired the growth of pre-establishedPDX-T1 xenograft tumors.

FIGS. 5A and 5B are graphs showing the shRNA counts in the in vivogrowth experimental sample versus the shRNA counts in the baselinesample (FIG. 5A) and the shRNA counts in the in vitro growthexperimental sample versus the shRNA counts in the baseline sample (FIG.5B).

FIG. 5C is a schematic of the criteria used to narrow the initial listof hits from the in vitro and the in vivo screens down to 46 candidategenes.

FIG. 5D is a list of 46 candidate genes determined from the in vitro andthe in vivo screens after filtering with the criteria shown in FIG. 5C.CDK19 is boxed.

FIG. 6A is a bar graph showing that TCGA breast cancer samples frompatients with the TNBC subtype are enriched in CDK19 copy numberamplifications or CDK19 mRNA upregulation compared to other subtypes.

FIG. 6B includes confocal immunofluorescent images of PDX-T1 stainedwith cytokeratin 8 (CK8) antibodies (first image from the left), CDK19antibodies (second image), and DAPI (third image). The composite imagecomposed from all three aforementioned images is shown on the far right(images are representative of three independent experiments).

FIGS. 7A and 7B are bar graphs showing that CDK19 targeting shRNAeffectively silences CDK19 in TNBC cells lines. Expression of CDK19 inMDA-MB231 (FIG. 7A) or MDA-MB468 (FIG. 7B) determined by RT-qPCR forcells transduced with control shRNA, shCDK19-1, and shCDK19-2.

FIG. 7C is a bar graph showing that CDK19 targeting shRNA effectivelysilences CDK19 in a TNBC PDX. Expression of CDK19 in PDX-T1 asdetermined by RT-qPCR for cells transduced with control shRNA,shCDK19-1, and shCDK19-2.

FIG. 7D includes images of tissue samples and representative images ofmouse lungs bearing PDX-T4 metastases. Lungs from mice with PDXstransduced with control shRNA (top row), shCDK19-1 (middle row), orshCDK19-2 (bottom row) are shown. Bright field images (left column) showgross lung morphology, FITC images (middle column) identify metastatictumor cells labeled with GFP, and Texas-Red images (right column)identify shRNA-transduced metastatic cells labeled with RFP.

FIG. 8A is a graph showing the flow cytometry analyses of TNBC (PDX-T1)using EpCAM and CD49f and the overlap of theEpCAM^(med/high)/CD10^(-/low) (1), EPCAM^(low/med)/CD10^(low/+) (3) andEpCAM⁻/CD10⁻ ( (2)) sub-populations.

FIG. 8B is a bar graph showing that the induction of CDK19 shRNA withdoxycycline effectively silences CDK19 in inducCDK19KD-PDX-Tl cells.Expression of CDK19 in control inducCDK19KD-PDX-Tl cells (black bar) anddoxycycline treated inducCDK19KD-PDX-Tl cells (gray bar) as determinedby RT-qPCR.

FIG. 8C shows that CDK19 knockdown effectively prevents the growth ofxenograft tumors in a limiting dilution assay.

FIG. 8D is a graph showing ELDA (Hu et al., Journal of Immunol. Methods347:70-78, 2009) analysis of the data from FIG. 8C to determine tumorinitiating frequencies in the doxycycline (Group +Dox) and controlgroups (Group NoDox). P-values as determined by the ELDA software.

FIG. 9 shows the amino acid sequence alignment showing 84% sequencehomology between CDK19 and CDK8. Amino acid positions are shown abovethe sequence. Alignment is performed using Clustal W method withMegAlign (DNAStar).

FIG. 10 is a table showing hallmark gene sets found enriched by GSEA ofthe genes upregulated or downregulated by either CDK19 knockdown or CDK8knockdown.

FIG. 11 is a graph showing that genome-wide H3K27Ac CHIP-Seq signalsacross all identified H3K27Ac peak regions are not significantlydifferent between the CDK19 knockdown, CDK8 knockdown, and controlsamples. Aggregate plots of normalized H3K27Ac CHIP-Seq signals acrossall H3K27Ac peak regions in the CDK19 knockdown (1), CDK8 knockdown (2)and control (3) samples (ns is P > 0.05, all samples n = 3, experimentsperformed three times).

FIGS. 12A and 12B show heat map of the expression of CDK19KD-EnhancerUP‘core’ genes (FIG. 12A) and CDK19KD-EnhancerDOWN ‘core’ genes (FIG.12B). Normalized expression of each gene in each biological replicate ofthe CDK19 knockdown and Control samples are shown.

FIGS. 13A-13D are graphs showing representative genes where CDK19knockdown leads to changes in H3K27Ac signals and corresponding changesin gene expression. Representative gene tracks depicting H3K27Ac signalsat the loci of select CDK19KD-EnhancerUP ‘core’ (FIGS. 13A and 13B) andCDK19KD-EnhancerDOWN ‘core’ genes (FIGS. 13C and 13D).

FIG. 13E is a heat map of the normalized gene expression of ELF3, ETV7,CHI3L2, and CRTAM across each of the three biological replicates incontrol and CDK19 knockdown samples.

FIGS. 14A and 14B are graphs showing that total body weights of micewere not significantly different between the mice fed doxycycline rodentfeed (doxycycline group) compared to the mice fed standard rodent feed(control group) in the inducCDK19KD-PDX-T1 (mean ± s.d., n = 5,experiments performed twice) (FIG. 14A) and inducCDK19KD-PDX-T3 (mean ±s.d., n = 5, experiment performed once) (FIG. 14B) tumor experiments.

FIG. 14C is a graph showing that total body weights of mice were notsignificantly different between the mice receiving oral gavage withCCT251921 compared to Vehicle (mean ± s.d., n = 5, experiment performedonce).

FIG. 15 is a table showing the pathological features and patientinformation for the patient derived xenograft tumors used in theexperiments.

FIGS. 16A-16D show a nucleic acid alignment of the 3′ UTR of CDK8 andCDK19. The underlined and bolded text indicates the overlapping regions.

FIG. 17 shows a nucleic acid alignment of the 5′ UTR of CDK8 and CDK19.The underlined and bolded text indicates the overlapping regions.

DETAILED DESCRIPTION OF THE INVENTION 1. Introduction - Cdk19 IsRequired for Triple-Negative Breast Cancer (TNBC) Growth

We have discovered that reducing expression or activity of CDK19 in TNBCcell lines or breast cancer patient derived xenografts in mice inhibitsgrowth and metastases of Triple Negative Breast Cancer (TNBC) tumors.See §4 below (Examples). We have also shown that the biologicalfunctions of CDK19 are distinct from those of its paralog, CDK8, andthat the CDK19-mediated effect on TNBC tumors is independent of CDK8activity. These data demonstrate that TNBC can be treated by agents thatinhibit CDK19 but do not inhibit CDK8, or agents that preferentiallyinhibit CDK19 compared to CDK8. The discovery that inhibition of CDK19is necessary and sufficient for inhibition of TNBC growth and metastasesis significant, in part, because of the potential advantages of CDK19 asa therapeutic target. Compared to other ubiquitous transcriptionalco-factors, such as CDK8, CDK9, and BRD4, CDK19 has more limited tissuedistribution, suggesting reduced toxicity and a broader therapeuticwindow for CDK19 inhibitors.

In addition to demonstrating that CDK19 knockdown had tumor growthinhibitory effects, CDK19 expression was also shown to be enriched intumor initiating cells, e.g., tumorigenic cells havingEpCAM^(med/high)/CD10^(-/low) expressions, compared to the lesstumorigenic cells, e.g., cells having EPCAM^(low/med)/CD10^(low/+)expressions (see, e.g., Example 4). Further studies also showed thatCDK19 knockdown significantly decreased tumor initiating frequencies(FIG. 8D). This discovery indicates that, compared to other agents,targeting CDK19 will result in a more pronounced and significant effecton highly tumorigenic (e.g., tumor initiating) cells. These discoveriesalso allow development of theranostic methods for identifying certainTNBC patients likely to respond to CDK19 targeted therapy.

2. Definitions 2.1 Triple-Negative Breast Cancer (TNBC)

Triple-negative breast cancer (TNBC) is a breast cancer subtypecharacterized by lack of expression of estrogen receptor (ER),progesterone receptor (PR), and human epidermal growth factor receptor 2(Her2). Receptor expression can be measured by immunohistochemicalstaining or other methods. TNBC is generally a diagnosed by exclusion.Widely used breast cancer therapies that target these receptors are noteffective against TNBC, making TNBC treatment particularly challenging.

2.2 Cyclic-Dependent Kinase 19 (CDK19)

Cyclic-Dependent Kinase 19 (CDK19) is described in Broude et al., Curr.Cancer Drug Targets 15:739, 2015 and Sato et al., Molecular Cell14:685-691, 2004. CDK19 belongs to a subset of the CDK family that isreportedly more associated with regulation of RNA polymerase II (RNAPII)transcription (see, e.g., Galbraith et al., Transcription 1: 4-12, 2010)than cell cycle progression. See UniProt entry NP_055891.1; Genbankentries AY028424 & AL603914. The mRNA sequences for CDK19 are alsodisclosed herein (e.g., SEQ ID NOs:12 -15).

2.3 Cyclic-Dependent Kinase 8 (CDK8)

CDK8 is a paralog of CDK19 with 84% amino acid sequence homology toCDK19. See FIG. 9 . CDK8 is described in Broude et al., Curr. CancerDrug Targets 15:739, 2015 and Sato et al., Molecular Cell 14:685-691,2004. See UniProt entry CAA59754.1; Genbank entries X85753 & AL590108.The mRNA sequences for CDK8 are also disclosed herein (e.g., SEQ IDNOs:16-18).

2.4 Agent

As used here, the term “agent” refers to a biological molecule (e.g.,nucleic acids, proteins, peptides, antibodies) or small organic molecule(e.g., having a molecular weight less than 1000, usually less than 500)that can reduce or inhibit the expression or activity of CDK19.

2.5 Inhibitors

As used herein, the term “inhibitor” as used in the context of CDK19,refers to a compound, composition or system that reduces the expressionor activity of CDK19. An agent may also selectively inhibit CDK19expression or activity over that of CDK8.

2.6 Knockdown

As used herein, the term “knock down” refers to a reduction in theexpression level of the CDK19 gene. Knocking down CDK19 gene expressionlevel may be achieved by reducing the amount of mRNA transcriptcorresponding to the gene, leading to a reduction in the expressionlevel of CDK19 protein. Knocking down CDK19 gene expression level mayalso be achieved by reducing the amount of CDK19 protein. An knockdownagent is an example of an inhibitor.

2.7 Knockout

As used herein, the term “knock out” refers to deleting all or a portionof the CDK19 gene in a cell, in a way that interferes with the functionof the CDK19 gene. For example, a knock out can be achieved by alteringthe CDK19 sequence. Those skilled in the art will readily appreciate howto use various genetic approaches, e.g., CRISPR/Cas systems, to knockoutthe CDK19 gene or a portion thereof. An knockout agent is an example ofan inhibitor.

2.8 Reduction Relative to a Reference Level

As used here, the terms “decrease,” “reduced,” “reduction,” and“decreasing” are all used herein to refer to a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 5%, at least about 10%, at least about 20%, or at least about 30%,or at least about 40%, or at least about 50%, or at least about 60%, orat least about 70%, or at least about 80%, or at least about 90% or upto and including a 100% decrease (i.e. absent level as compared to areference sample), or any decrease between 10-100% as compared to areference level.

2.9 Nucleic Acids

As used herein, the terms “polynucleotide,” “nucleic acid,” and“oligonucleotide” are used interchangeably and refer to a polymeric formof nucleotides of any length, either deoxyribonucleotides orribonucleotides or analogs thereof. A polynucleotide can comprisemodified nucleotides, such as methylated nucleotides and nucleotideanalogs, or otherwise be modified by art-known methods to render thepolynucleotide resistant to nucleases, improve delivery of thepolynucleotide to target cells or tissues, improve stability, reducedegradation, improve tissue distribution or to impart other advantageousproperties. For example, the DNA or RNA polynucleotide may include oneor more modifications on the oligonucleotide backbone (e.g., aphosphorothioate modification), the sugar (e.g., a locked sugar), or thenucleobase. If present, modifications to the nucleotide structure can beimparted before or after assembly of the oligonucleotide. The sequenceof nucleotides can be interrupted by non-nucleotide components. Anoligonucleotide can be further modified after polymerization, such as byconjugation with a label component, a targeting component, or othercomponent. Polynucleotides may be double-stranded or single-strandedmolecules. Furthermore, in order to improve the oligonucleotidedelivery, the DNA or RNA oligonucleotide may be packaged into a lipidmolecule (e.g., lipid nanoparticles) or be conjugated to acell-penetrating peptide.

2.10 Treatment

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment,” as used herein, can includetreatment resulting in inhibiting the disease, i.e., arresting itsdevelopment; and relieving the disease, i.e., causing regression of thedisease. For example, in the case of cancer, a response to treatment caninclude a reduction in cachexia, increase in survival time, elongationin time to tumor progression, reduction in tumor mass, reduction intumor burden and/or a prolongation in time to tumor metastasis, time totumor recurrence, tumor response, complete response, partial response,stable disease, progressive disease, progression free survival, overallsurvival, each as measured by standards set by the National CancerInstitute and the U.S. Food and Drug Administration for the approval ofnew drugs and/or described in Eisenhauer, EA1, et al. “New responseevaluation criteria in solid tumours: revised RECIST guideline (version1.1).” European journal of cancer 45.2 (2009): 228-247.

2.11 Administration

As used herein, the term “administering” or “administration” includesany route of introducing or delivering an agent that inhibits theexpression or activity of CDK19 to the subject diagnosed with TNBC.Administration can be carried out by any route suitable for the deliveryof the agent. Thus, delivery routes can include, e.g., intravenous,intramuscular, intraperitoneal, or subcutaneous deliver. In someembodiments, the agent is administered directly to the tumor, e.g., byinjection into the tumor.

2.12 Therapeutically Effective Dose

As used here, the term “therapeutically effective amount” refers to anamount, e.g., pharmaceutical dose, effective in inducing a desiredbiological effect in a subject or patient or in treating a patienthaving TNBC described herein. The term “therapeutically effectiveamount” refers to an amount of an active agent being administered thatwill treat to some extent a disease, disorder, or condition, e.g., TNBC,relieve one or more of the symptoms of the disease being treated, and/orthat amount that will prevent, to some extent, one or more of thesymptoms of the disease that the subject being treated has or is at riskof developing. For example, for a given parameter (e.g., tumor volume,tumor diameter, metastases, etc.), a therapeutically effective amountwill show an increase or decrease of therapeutic effect of at least 5%,10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or atleast 1-fold, 2-fold, or 3-fold. A therapeutically effective dose isusually delivered over a course of therapy that may extend for a periodof days, weeks, or months. A therapeutically effective dose of an agentmay be taken alone or in combination with other therapeutic agents. Insome cases, a therapeutically effective amount of a CDK19 inhibitor isam amount sufficient to effect a partial response in a patient with TNBC(e.g., a greater than 20% reduction, sometimes a greater than 30%reduction, in the measurable diameter of lesions).

2.13 Patient or Subject

A “patient” or “subject,” as used herein, is intended to include eithera human or non-human animal, preferably a mammal, e.g., non-humanprimate. Most preferably, the subject or patient is a human.

2.14 Antisense Strand

A “antisense strand” refers to the strand of a double stranded RNAiagent (siRNA or shRNA) which includes a region that is complementary orsubstantially complementary to a target sequence (e.g., a human CDK8 orCDK19 mRNA including a 5′ UTR, exons of an open reading frame (ORF), ora 3′ UTR). Where the region of “complementarity” or “substantiallycomplementary” need not be fully complementary to the target sequenceand may have sequence % identity or % similarity of least 70%, 75%, 80%,85%, 90%, 95%, or 100%.

2.15 Sense Strand

A “sense strand,” as used herein, refers to the strand of a RNAi agent(siRNA or shRNA) that includes a region that is complementary orsubstantially complementary to a region of the antisense strand.

3. Methods of Treatment

In one approach the invention provides a method of treating a patientdiagnosed with triple-negative breast cancer (TNBC), comprisingadministering a therapeutically effective dose of an agent that inhibitsexpression or activity of cyclin-dependent kinase 19 (CDK19). In someembodiments, the treatment results in an at least 10% reduction in tumorvolume within 6 month of initiating therapy.

In one approach the invention provides a method of treating a patientdiagnosed with triple-negative breast cancer (TNBC), wherein the canceris characterized by a tumor comprising EpCAM^(med/high)/CD10^(-/low)epithelial cells, the method comprising administering a therapeuticallyeffective dose of an agent that inhibits cyclin-dependent kinase 19(CDK19) expression or activity, wherein the treatment results in areduction of the ratio of cells having a medium to high expression levelof EpCAM and a low expression level of CD10 to normal cells in thetumor. In some embodiments, the method includes the step of detectingEpCAM^(med/high)/CD10^(-/low) epithelial cells in a tissue sample fromthe patient prior to or after initiating therapy.

To determine the phenotype of a tumor or to assess treatment prognosis,a biopsy may be obtained from the patient diagnosed with TNBC. A biopsymay be a needle biopsy, or may be a liquid biopsy be obtained from bloodvessels and/or lymph nodes that supply the breast, e.g., internalmammary arteries, lateral thoracic arteries, thoracoacromial arteries,axillary lymph nodes.

As described in §4, below, CD10 and EpCAM biomarkers identify threedistinct sub-populations of Tumor Initiating Cells (TICs) in TNBC.EpCAM^(med/high)/CD10^(-/low), EPCAM^(low/med)/CD10^(low/+), andEpCAM⁻/CD10. The phenotype of cancer cells in a TNBC patient can bedetermined using art-known methods. In one approach a tissue is obtainedfrom the patient and the cell phenotype determined usingimmunohistochemistry, mass spectrometry analysis, fluorescence activatedcell sorting (FACS) or other methods. The cell phenotype can be assignedrelative to standard values characteristic of health or canceroustissue. In one approach the ratio of EpCAM^(med/high)/CD10^(-/low) cellsto normal breast epithelial cells is determined prior to initiation oftreatment to assess the likely response of the patient to CDK19 targetedtherapy. In one approach a change in the ratio of EpCAM^(med/high)/CD10^(-/low) cells to normal cells, or a change in the quantity ofEpCAM^(med/high)/CD10^(-/low) cells per volume tissue is detected afterinitiation of treatment.

In one approach the invention provides a method for reducing metastasisof TNBC in a patient, the method comprising administering atherapeutically effective dose of an agent that inhibits expression oractivity of CDK19

In some embodiments, methods of the invention may be used to treatinflammatory TNBCs or TNBCs that are chemo-resistant. In otherembodiments, the methods of the invention may be used to slow down orprevent the metastasis of TNBCs. In further embodiments, the methodsdescribed herein that target the CDK19 gene or its corresponding proteinmay further modulate clinically relevant TNBC pathways regulated byCDK19, such as P53 signaling, KRAS signaling, androgen response, NOTCHsignaling, TGF BETA signaling, and IL6-JAK-STAT3 signaling (FIG. 3B),and make them more therapeutically susceptible to cancer treatments.

3.1 Therapeutic Agents (Inhibitors) 3.1.1. Polynucleotides

As demonstrated in the examples, the CDK19 gene is essential for thegrowth of TNBC. Methods of treating TNBC in a subject as describedherein may be accomplished by administering a polynucleotide (e.g.,oligonucleotide) to the subject to decrease or inhibit the expression ofthe CDK19 gene. In some embodiments, the polynucleotide may be, forexample, a DNA oligonucleotide or an RNA oligonucleotide. In otherembodiments, the oligonucleotide may be used in a CRISPR/Cas system. Anoligonucleotide that inhibits or decreases the expression of the CDK19gene may knock out or knock down the CDK19 gene (e.g., the CDK19 gene ina TNBC cell) in the subject.

In some embodiments, the oligonucleotide may be an shRNA or an miRNA. Insome embodiments, the oligonucleotide may mediate an RNase H-dependentcleavage of the mRNA transcript of the CDK19 gene. In other embodiments,the oligonucleotide may be used in a CRISPR/Cas system.

In some embodiments, the mRNA transcript of the CDK19 gene may betargeted for cleavage and degradation. Different portions of the mRNAtranscript may be targeted to decrease or inhibit the expression of theCDK19 gene. In some embodiments, a DNA oligonucleotide may be used totarget the mRNA transcript and form a DNA:RNA duplex with the mRNAtranscript. The duplex may then be recognized and the mRNA cleaved byspecific proteins in the cell. In other embodiments, an RNAoligonucleotide may be used to target the mRNA transcript of the CDK19gene.

3.1.1.1. shRNA

A short hairpin RNA or small hairpin RNA (shRNA) is an artificial RNAmolecule with a hairpin turn that can be used to silence target geneexpression via the small interfering RNA (siRNA) it produced in cells.See, e.g., Fire et. al., Nature 391:806-811, 1998; Elbashir et. Al.,Nature 411:494-498, 2001; Chakraborty et al. Mol Ther Nucleic Acids8:132-143, 2017;, Bouard et al., Br. J. Pharmacol. 157:153-165, 2009.Expression of shRNA in cells is typically accomplished by delivery ofplasmids or through viral or bacterial vectors. Suitable bacterialvectors include but not limited to adeno-associated viruses (AAVs),adenoviruses, and lentiviruses. Once the vector has integrated into thehost genome, the shRNA is then transcribed in the nucleus by polymeraseII or polymerase III depending on the promoter choice. The resultingpre-shRNA is exported from the nucleus and then processed by Dicer andloaded into the RNA-induced silencing complex (RISC). The sense strandis degraded by RISC and the antisense strand directs RISC to an mRNAthat has a complementary sequence. A protein called Ago2 in the RISCthen cleaves the mRNA, or in some cases, represses translation of themRNA, thus, leading to its destruction and an eventual reduction in theprotein encoded by the mRNA. Thus, the shRNA leads to targeted genesilencing. shRNA is an advantageous mediator of siRNA in that it hasrelatively low rate of degradation and tu rnover.

In some embodiments, the methods described herein include treating TNBCin a subject using an shRNA. The methods may include administering tothe subject a therapeutically effective amount of a vector, wherein thevector includes a polynucleotide encoding an shRNA capable ofhybridizing to a portion of an mRNA transcript of the CDK19 gene. Insome embodiments, the vector may also include appropriate expressioncontrol elements known in the art, including, e.g., promoters (e.g.,tissue specific promoters), enhancers, and transcription terminators.Once the vector is delivered to the TNBC cell, the shRNA may beintegrated into the cell’s genome and undergo downstream processing byDicer and RISC (described in detail further herein) to eventuallyhybridize to the mRNA transcript of the CDK19 gene, leading to mRNAcleavage and degradation. In some embodiments, the shRNA may include anucleic acid sequence that has at least 85% sequence identity to thesequence of GCGAGAATTGAAGTACCTTAA (SEQ ID NO: 1) or the sequence ofACCAGCAAATATCCTAGTAAT (SEQ ID NO: 2). In particular embodiments, theshRNA may target the amino acids at the N-terminus of an mRNA transcriptof the CDK19 gene. In other embodiments, the shRNA may target the aminoacids at an internal region of an mRNA transcript of the CDK19 gene.

As demonstrated in the Examples, e.g., FIGS. 1G-1J, both shRNAs(GCGAGAATTGAAGTACCTTAA (SEQ ID NO: 1) and ACCAGCAAATATCCTAGTAAT (SEQ IDNO: 2)) targeted against the CDK19 gene were able to knockdown the gene,which led to a significant reduction in the percentage of RFP positivecells in tumors from all three TNBC PDXs. Further, CDK19 knockdown alsoinhibited the growth of an aggressive PDX obtained from the brainmetastasis of a patient with a chemotherapy-resistant inflammatorybreast cancer (FIG. 1J), which was known to be aggressive, difficult totreat, and associated with extremely poor prognoses. In addition toinhibiting tumor growth, shRNAs also inhibited the lung metastases ofthese tumors in mice (FIG. 1L).

In some embodiments, an shRNA targeted against the CDK19 gene may haveat least 85% sequence identity (e.g., 87%, 89%, 91%, 93%, 95%, 97%, or99% sequence identity) to GCGAGAATTGAAGTACCTTAA (SEQ ID NO: 1). In otherembodiments, an shRNA targeted against the CDK19 gene may have at least85% sequence identity (e.g., 87%, 89%, 91%, 93%, 95%, 97%, or 99%sequence identity) to ACCAGCAAATATCCTAGTAAT (SEQ ID NO: 2). In otherembodiments, an shRNA targeted against the CDK19 gene may have at least85% sequence identity (e.g., 87%, 89%, 91%, 93%, 95%, 97%, or 99%sequence identity) to GCTTGTAGAGAGATTGTACTT (SEQ ID NO: 3). In someembodiments, an shRNA targeted against the CDK19 gene may have at least85% sequence identity (e.g., 87%, 89%, 91%, 93%, 95%, 97%, or 99%sequence identity) to GAGGACTGATAGTTCTTCTTT (SEQ ID NO: 4). In otherembodiments, an shRNA targeted against the CDK19 gene may have at least85% sequence identity (e.g., 87%, 89%, 91%, 93%, 95%, 97%, or 99%sequence identity) to GATATTAGAAAGATGCCAGAA (SEQ ID NO: 5). In otherembodiments, an shRNA targeted against the CDK19 gene may have at least85% sequence identity (e.g., 87%, 89%, 91%, 93%, 95%, 97%, or 99%sequence identity) to GCCAACAGTAGCCTCATAAAG (SEQ ID NO: 6). In otherembodiments, an shRNA targeted against the CDK19 gene may have at least85% sequence identity (e.g., 87%, 89%, 91%, 93%, 95%, 97%, or 99%sequence identity) to CGTTCGTATTTATCTAGTTTC (SEQ ID NO: 7). In otherembodiments, an shRNA targeted against the CDK19 gene may have at least85% sequence identity (e.g., 87%, 89%, 91%, 93%, 95%, 97%, or 99%sequence identity) to GCATGACTTGTGGCATATTAT (SEQ ID NO: 8). In otherembodiments, an shRNA targeted against the CDK19 gene may have at least85% sequence identity (e.g., 87%, 89%, 91%, 93%, 95%, 97%, or 99%sequence identity) to GCTTGTAGAGAGATTGCACTT (SEQ ID NO: 9). In otherembodiments, an shRNA targeted against the CDK19 gene may have at least85% sequence identity (e.g., 87%, 89%, 91%, 93%, 95%, 97%, or 99%sequence identity) to AGGACTGATAGCTCTTCTTTA (SEQ ID NO: 10). In yetother embodiments, an shRNA targeted against the CDK19 gene may have atleast 85% sequence identity (e.g., 87%, 89%, 91%, 93%, 95%, 97%, or 99%sequence identity) to GTATGGCTGCTGTTTGATTAT (SEQ ID NO: 11). One ofskill in the art has the knowledge and capability to design shRNAs thattarget different portions of the CDK19 gene (e.g., the 5′ UTR region orthe 3′ UTR region) to achieve the desired reduction in expression of thegene. For example, available tools for designing shRNAs include, e.g.,Project Insilico, Genomics and Bioinformatics Group, LMP, CCR, NIH. Insome embodiments, an shRNA may be designed to knockout the CDK19 gene.

CDK8 and CDK19 shRNA

There are a number of structural elements that can affect shRNAefficacy. For specific RNAi knockdown of a desired target gene an shRNAcan be designed in consideration of its multiple structural elements.Generally, an shRNA should be about 80 nucleotides in length anddesigned (from 5′ to 3′) to comprise of the following structuralelements to make the hairpin structure of the shRNA: (1) a sense strand(e.g., upper stem); (2) followed by a hairpin loop; (3) an antisensestrand (e.g., lower stem or guide strand) that has perfect or nearperfect complementary to the target mRNA and is antisense to the targetmRNA; (4-5) two cleavage motifs such as, “U” or “UH” at the firstposition of the guide strand, and “UUC” or “CUUC” at the tail region ofthe guide strand; and (6) arbitrary spacer nucleotides of about twonucleotides in length between the first nucleotide of guide strand “U”motif and the hairpin loop, and between the last nucleotide of the sensestrand and the hairpin loop. The sense strand and antisense strand,making up the stem, may be designed to consist of a range from about 19to 29 nucleotides in length, which will form the stem. The loopstructure may be designed to consist of a range about 2 to 15nucleotides in length, and preferably free of any internal secondarystructure. Some examples of sequences that may be used for making thehairpin loop, include but are not limited to, a nine nucleotide loopcomprising the sequence (TTCAAGAGA), and a seven nucleotide loopcomprising the sequence (TCAAGAG). Other design strategies can be foundin the relevant disclosure of Ros XB-D, Gu S. Guidelines for the optimaldesign of miRNA-based shRNAs. Methods (San Diego, Calif)2016;103:157-166, which is herein incorporated by reference in itsentirety for all purposes. There are also several design programsavailable such as, The RNAi Consortium software from The BroadInstitute, which is made available through Sigma-Aldrich andThermo-Fisher Scientific.

The specificity of the target sequence should also be considered, asmany mRNAs can share similar sequences. Care should be taken inselecting target sequence that has low sequence homology to other genesin the genome to allow for gene-specific knockdown. Where a gene hasmultiple forms, to achieve complete knockdown of gene expression, shRNAshould target sequences shared among all isoforms of the target mRNA.

An alignment of CDK19 and CDK8 mRNA sequences can identify not identicalor low percent identity or similarity nucleotide sequence regions whichcan be used to design shRNAs that have a preference to target to CDK19mRNA but not CDK8, see for example the 3′ UTR and 5′ UTR alignments inFIG. 16 and FIG. 17 .

In some embodiments, shRNA that targets a CDK19 mRNA transcript, and notof CDK8 mRNA transcript can be designed. In one approach the mRNAsequences for human CDK19 and CDK8 from National Center forBiotechnology Information (NCBI, found at Pubmed.gov) and an alignmentiis performed (e.g., with pairwise alignment program such as, LALIGN). Aregion of about 19 to 29 contiguous nucleotides (e.g., 19-20, 19-21,19-22, 19-23, 19-24, 19-25, 19-26, 19-27, 19-28, or 19-29) in length isselected based on low sequence identity (e.g., less than 75%, identity,sometimes less than 70% identity, sometimes less than 60% identity. Insome embodiments the 19 to 29 nt region has very low (e.g., less than40%, less than 30% or less than 20% or sequence identity. The contiguoussequence can be in a protein coding region, the 5′-UTR, the 3′-UTR, orspan two regions.

In one embodiment, target-specific knockdown of CDK19 can beaccomplished by designing an shRNA with a guide strand that iscomplementary of the 3′ UTR region of CDK19 (SEQ ID NO:42) and has lowor no homology to the 3′UTR of CDK8 (SEQ ID NO:44). The guide strand maybe 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides in length.Some exemplary sequence regions that may be used to design a CDK19shRNA, include but are not limited to, CTCCAGCTCCCGTTGGGCCAGGCCAGCCC(SEQ ID NO: 20), AGCCCAGAGCACA GGCTCCAGCAATATGT (SEQ ID NO: 21),CTGCATTGAAAAGAACCAAAAAAATGCAA (SEQ ID NO: 22),ACTATGATGCCATTTCTATCTAAAACTCA (SEQ ID NO: 23), TACACATGGGAGGAAAACCTTATATACTG (SEQ ID NO: 24), AGCATTGTGCAGGACTGATAGCTCTTCTT (SEQ IDNO: 25), TATTGACTTAAAGAAGATTCTTGTGAAGT (SEQ ID NO: 26), TTCCCCTATCTCAGCACCCCTTCCCTGCA (SEQ ID NO: 27), TGTGTTCCATTGTGACTTCTCTGATAAAG (SEQ ID NO:28), CGTCTGATCTAATCCCAGCACTTCTGTAA (SEQ ID NO: 29), or CCTTCAGCATTTCTTTGAAGGATTCTATC (SEQ ID NO: 30). One of ordinary skill guided by thisdisclosure understands that other low homology sequence regions in the‘3 UTR could also be used. See, for example, FIGS. 16A-D the lowhomology sequence regions from (1-1186) and (2418-4570). In oneembodiment, the shRNA may be designed to be targeted to upstream ofCDK19, downstream of CDK19, or in the exons of CDK19. In some cases theexpression of the CDK19 shRNA results in knockdown of CDK19 at leastabout 25%, 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. Inanother embodiment the expression of the CDK19 shRNA can preferentiallyknockdown CDK19 compared to CDK8.

To make shRNAs that preferentially target CDK19 one would identify aunique region of CDK19, a region that does not have significant homologyto other CDKs (e.g., CDK8) or other mRNAs in the genome. One would usethis sequence to make a guide strand that is antisense to this targetand comprises 19 to 29 nucleotides in length. To make the expressioncassette one would add an appropriate promoter such as a pol II or polIII promotor at the beginning of the cassette, followed by thecomplementary sense strand (e.g., complementary to the targeting guidestrand), which is them followed by the loop structure of about 2 to 15nucleotides in length. In addition, the two Ago cleavage motifs, “U” or“UH” should be included at the first position of the guide strand, and“UUC” or “CUUC” at the tail region of the guide strand along to 1-2spacer nucleotides at the end of the loop structure. See, for example USApplication No. US2008/0293142 and Ros XB-D, Gu S. Guidelines for theoptimal design of miRNA-based shRNAs. Methods (San Diego, Calif)2016;103:157-166, which is herein incorporated by reference in itsentirety for all purposes.

In another embodiment, target-specific knockdown of CDK8 can beperformed by using an shRNA with a guide strand that comprises acomplementary to the 5′UTR of CDK8 (SEQ ID NO: 43) and has low or nohomology to the 5′ UTR of CDK19 (SEQ ID NO:41). The guide strand may be19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29, nucleotides in length.Some exemplary sequences that may be used to design a CDK8 shRNA includebut are not limited to, TGGCCGCCCCGCCGCTCCCGCCGCAGCAG (SEQ ID NO: 31),GAGCAGAACGCGCGGCCGGAGA GAGCGGC (SEQ ID NO: 32),GGAGCCGGCGCCCAGGGAGCCCGCGGGGA (SEQ ID NO: 33),CAAGGGCAGAGACACCGCTCCCCACCCCC (SEQ ID NO:34),AGCCCTCGTCCCTCGGCTCTCCTTCGCCG (SEQ ID NO: 35),GGGGATCCTCCCCGTTCCTCCACCCCCGG (SEQ ID NO: 36), CCGGCCTCTGCCCCGCCGTCCCCCTGGAT (SEQ ID NO: 37), GTCCCTGGCGCTTTCGCGGGGCCTCCTCC (SEQID NO: 38), TGCTCTTGCCGCATCAGTCGGGCTGGTGC (SEQ ID NO: 39), orTGCGGCCGGCGGGCGTAGAGC GGGCGGGT (SEQ ID NO: 40). One of ordinary skill inthe art would understand that other low homology sequence regions in the‘5 UTR could also be used. See, for example, FIG. 17 the low homologysequence regions from (1-33) or (223 -504). In another embodiment theshRNA may be designed to be targeted to upstream of CDK8, downstream ofCDK8, or in the exons of CDK8. In some cases, the expression of the CDK8shRNA can result in a knockdown of CDK8 at least about 25%, 50%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%..

To make shRNAs that preferentially target CDK8 one would identify aunique region of CDK8, a region that does not have significant homologyto other CDKs (e.g., CDK19) or other mRNAs in the genome. One would usethis sequence to make a guide strand that is antisense to this targetand comprises 19 to 29 nucleotides in length. To make the expressioncassette one would add an appropriate promoter such as a pol II or polIII promotor at the beginning of the cassette, followed by thecomplementary sense strand (e.g., complementary to the targeting guidestrand), which is them followed by the loop structure of about 2 to 15nucleotides in length. In addition, the two Ago cleavage motifs, “U” or“UH” should be included at the first position of the guide strand, and“UUC” or “CUUC” at the tail region of the guide strand along to 1-2spacer nucleotides at the end of the loop structure. See, for example USApplication No. US2008/0293142 and Ros XB-D, Gu S. Guidelines for theoptimal design of miRNA-based shRNAs. Methods (San Diego, Calif)2016;103:157-166, which is herein incorporated by reference in itsentirety for all purposes.

The specificity or knockdown level of an shRNA or siRNA can be confirmedusing real-time PCR analysis for mRNA level or ELISA assay for theprotein level. Experimental controls may be run in parallel to assessknockdown. Some examples of experimental controls that may be used,include but are not limited to, a mock-infected or mock-transfectedsample, an empty vector, an shRNA encoding a scrambled target or seedregion, an shRNA targeting another gene entirely such as, housekeepinggenes GAPDH or Actin, or a GFP positive control.

To determine if an siRNA or shRNA (e.g., RNAi agent) preferentiallytargets CDK19 over CDK8 one can transfect or transduce the shRNA orsiRNA tagged to marker such as GFP in a cell line or other expressionsystem, select the GFP positive cells (e.g. transformed cells), anddetermine the level of CDK19 knockdown relative to CDK19 expression inthe cell system without transfection or transduction with the RNAiagent. In some embodiments, the expression of RNA is measured. In otherembodiments, the expression of the protein is measured. In one example,mRNA may be measured by any PCR-based assay known in the art (e.g.,RT-PCR or qRT-PCR or the like). In one example, the protein level may bemeasured by an immunoassay (e.g., ELISA assay or any antibody-basedmethod known in the art).

In some embodiments, a targeting CDK19 shRNA or siRNA results in CDK19expression less than about 30% and CDK8 greater than about 70% relativeto a system without transfection or transduction. In some otherembodiments, a targeting CDK19 shRNA or siRNA results in CDK19expression at less than about 50% and CDK8 greater than about 95%. Insome embodiments, a targeting CDK19 shRNA or siRNA results in CDK19expression less than about 5% and CDK8 greater than about 80%. In someembodiments, a targeting CDK19 shRNA or siRNA results in CDK19expression less than about 1% and CDK8 greater than about 60%. In someembodiments, a targeting CDK19 shRNA or siRNA results in CDK19expression at less than about 0.5% and CDK8 greater than about 90%. Insome embodiments, a targeting CDK19 shRNA results in CDK19 expression atabout 0% and CDK8 at about 100% relative to a system withouttransfection or transduction. In some embodiments, the expression of RNAis measured. In other embodiments, the expression of the protein ismeasured.

CDK8 and CDK19 siRNA

The present disclosure also provides siRNA-based therapeutics forinhibiting expression of CDK8 and CDK19 in a patient withtriple-negative breast cancer. The double stranded RNAi therapeuticincludes a sense strand complementary to an antisense strand. The senseor antisense strands of the siRNA may be about 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. Theantisense strand of the siRNA-based therapeutic includes a regioncomplementary to a part of an mRNA encoding CDK8 or CDK19. Additionalmethods to make therapeutic siRNA can be found in U.S. Pat No.US9399775, which is incorporated by reference in its entirety for allpurposes.

In some cases, the expression of CDK19 siRNA may result in a knockdownof CDK19 at least about 25%, 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100%. In another embodiment, the expression of CDK19 siRNAmay preferentially knockdown CDK19 compared to CDK8. In some cases, theexpression of CDK8 siRNA may result in a knockdown of CDK8 at leastabout 25%, 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

In a preferred embodiment, CDK19 siRNA may result in a knockdown ofCDK19 at least about 25%, 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% and CDK8 at least about 10%, 14%, 15%, 16%, 17%, 18%, 19%,20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%.

shRNA and siRNA Delivery

Depending on whether transient or stable expression is desired one canselect an appropriate delivery vector. Examples of delivery vectors thatmay be used with the present disclosure are viral vectors, plasmids,exosomes, liposomes, bacterial vectors, or nanoparticles. The presentdisclosure also provides for delivery by any means known in the art.

For targeted delivery to triple-negative breast cancer cells, oneskilled in the art would appreciate that delivery vectors may begenetically modified to target a specific cell type or to tissue type.To make a targeted delivery vector or plasmid one can identify a uniquemolecule expressed or associated with a triple-negative breast cancer(e.g., receptor, protein, glycoprotein, or combination thereof) and thencreate a delivery vector or plasmid that harbors or expresses thesemarkers, preferably on the outside of the delivery vector or plasmid(e.g., cytosol facing). In addition, depending on the requiredtherapeutic duration a viral delivery vector can be genetically modifiedto be continuously replicating, replication-defective, or conditionallyreplicating as described in, Sliva K, Schnierle BS. Selective genesilencing by viral delivery of short hairpin RNA. Virology Journal.2010.

In one embodiment, the CDK8 or CDK19 shRNA or siRNA can be delivered byan adenovirus vector. Adenoviruses non-enveloped viruses with anucleocapsid and a linear dsDNA genome. While they are able to replicatein the nucleus of mammalian cells, they do not efficiently integrateinto the host’s genome and therefore pose only minimal risks ofinsertional mutagenesis but are inadequate for long-term therapy.

In another embodiment, the CDK8 or CDK19 shRNA or siRNA can be deliveredby an adeno-associated viral vector (AAV). AAV is one of the smallestviruses and belongs to the genus Dependovirus. It has a small,single-stranded DNA genome and can accommodate about eight individualshRNA. AAV permits entry retargeting, allowing delivery of the shRNA tospecific cell or tissue types. In a further embodiment, the presentdisclosure provides for a modified AAV that is targeted for delivery toa triple-negative breast cancer cell or tissue type.

In another embodiment, the CDK8 or CDK19 shRNA or siRNA can be deliveredby a retrovirus vector. A retrovirus is a single-stranded RNA virus thatbelongs to the family of Retroviridae and replicate through adouble-stranded DNA intermediate. They can integrate into a host’sgenome thereby allowing long-term expression of a shRNA. The Env proteinplays a central role in targeting retrovirus to a target cell. In afurther embodiment, the present disclosure provides for a retrovirusvector with a modified env gene or its protein product for delivery to atriple-negative breast cancer cell or tissue type. In a furtherembodiment, the present disclosure provides for delivery of CDK8 orCDK19 shRNA of siRNA using a retrovirus vector with protease-activatedEnv proteins.

In another embodiment, the CDK8 or CDK19 shRNA or siRNA can be deliveredby a lentivirus vector. Lentivirus is a subclass of retrovirus in thegenus Lentivirinae which can accommodate large amounts of DNA. For someapplications, it may be preferable to use a lentivirus vector engineeredto be “self-inactivating” known as “SIN” vectors. In a furtherembodiment, the present disclosure provides for delivery of a CDK8 orCDK19 shRNA by a lentivirus vector with a modified env gene or itsprotein product for delivery to a triple-negative breast cancer cell ortissue type.

In another embodiment, the shRNA or siRNA can be delivered by ananoparticle. Examples of nanoparticles that can be use with the presentdisclosure, include but are not limited to, exosomes, liposomes, organicnanoparticles, or inorganic nanoparticles. Other non-limiting examplesof nanoparticles include, but are not limited to, e.g., those providedin Hong, Cheol Am, and Yoon Sung Nam. “Functional Nanostructures forEffective Delivery of Small Interfering RNA Therapeutics.” Theranostics4.12 (2014): 1211-1232. PMC. Web. 13 Sept. 2018, which is herebyincorporated by reference in its entirety for all purposes. In someembodiments, the delivery of the shRNA or siRNA is mediated by receptor,protein, glycoprotein or combination thereof present or specific totriple-negative breast cancer cells.

In some embodiments, the siRNA CDK19 therapeutic is administered in asolution. The siRNA may be administered in an unbuffered solution. Inone embodiment, the siRNA is administered in water. In otherembodiments, the siRNA is administered with a buffer solution, such asan acetate buffer, a citrate buffer, a prolamine buffer, a carbonatebuffer, or a phosphate buffer or any combination thereof. In someembodiments, the buffer solution is phosphate buffered saline.

3.1.1.2. Rnase H-Mediated MRNA Degradation/Antisense

RNase H-dependent antisense oligonucleotides (ASOs) are single-stranded,chemically modified oligonucleotides that bind to complementarysequences in target mRNAs and reduce gene expression both by RNaseH-mediated cleavage of the target RNA and by inhibition of translationby steric blockade of ribosomes.

RNase H is an endonuclease enzyme that catalyzes the cleavage of RNA inan RNA:DNA duplex. The most well studied endogenous function for thisenzyme is the removal of Okazaki fragments (small RNAs) used to primethe DNA duplication during cell division. In some embodiments, to targetthe mRNA transcript of the CDK19 gene for degradation, a nucleic acid(e.g., DNA oligonucleotide) capable of hybridizing to a portion of themRNA may be administered to the subject. Once inside the cell (e.g., aTNBC cell), the DNA oligonucleotide base pairs with its targeted mRNAtranscript. RNase H may bind to the resulting duplex and cleave the mRNAtranscript at one or more places. The DNA oligonucleotide may furtherbind to other mRNA transcripts to target them for RNase H degradation.Thus, the expression of the CDK19 gene may be greatly reduced in asubject with TNBC.

The DNA oligonucleotide capable of hybridizing to an mRNA transcript ofa CDK19 gene may contain, e.g., between 10 and 30 nucleotides (e.g., 10,12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 nucleotides). In someembodiments, the DNA oligonucleotide may have 100% complementarity tothe portion of the mRNA transcript it binds. In other embodiments, theDNA oligonucleotide may have less than 100% complementarity (e.g., 95%,90%, 85%, 80%, 75%, or 70% complementarity) to the portion of the mRNAtranscript it binds, but can still form a stable RNA:DNA duplex for theRNase H to cleave the mRNA transcript. The DNA oligonucleotide may bindto the 5′ UTR or the 3′ UTR of the mRNA transcript of the CDK19 gene.

Further, the DNA oligonucleotide capable of hybridizing to an mRNAtranscript of a CDK19 gene may contain modified nucleotides at the 5′end and the 3′ end. The modified nucleotides at the termini may functionto protect the internal portion of the DNA oligonucleotide from nucleasedegradation and to increase the binding affinity for the target mRNAtranscript. In some embodiments, the modified nucleotides at the terminimay include a modified nucleobase (e.g., 5-methylcytosine) and/or amodified sugar (e.g., a locked sugar). In some embodiments, 3-5nucleotides at each of the 5′ and 3′ ends of the DNA oligonucleotide maybe modified.

3.1.1.3. miRNA

A microRNA (miRNA) is a small non-coding RNA molecule that functions inRNA silencing and post-transcriptional regulation of gene expression.miRNAs base pair with complementary sequences within the mRNAtranscript. As a result, the mRNA transcript may be silenced by one ormore of the mechanisms such as cleavage of the mRNA strand,destabilization of the mRNA through shortening of its poly(A) tail, anddecrease translation efficiency of the mRNA transcript into proteins byribosomes. In some embodiments, miRNAs resemble the siRNAs of the shRNApathway, except that miRNAs derive from regions of RNA transcripts thatfold back on themselves to form short hairpins, which are also calledpri-miRNA. Once transcribed as pri-miRNA, the hairpins are cleaved outof the primary transcript in the nucleus by an enzyme called Drosha. Thehairpins, or pre-miRNA, are then exported from the nucleus into thecytosol. In the cytosol, the loop of the hairpin is cleaved off by anenzyme called Dicer. The resulting product is now a double strand RNAwith overhangs at the 3′ end, which is then incorporated into RISC. Oncein the RISC, the second strand is discarded and the miRNA that is now inthe RISC is a mature miRNA, which binds to mRNAs that have complementarysequences.

The difference between miRNAs and siRNAs from the shRNA pathway is thatbase pairing with miRNAs comes from the 5′ end of the miRNA, which isalso referred to as the seed sequence. Since the seed sequence is short,each miRNA may target many more mRNA transcript. In some embodiments, anmiRNA targeting the CDK19 gene may be used in methods described herein.

3.1.2. Crispr/Cas System

In some embodiments, the knocking out or knocking down of the CDK19 geneis performed using a gene editing system such as the CRISPR/Cas system.See Sanders and Joung, Nature Biotechnol 32:347-355, 2014, Huang et al.,J Cell Physiol 10:1-17, 2017 and Mitsunobu et al., Trends Biotechnol17:30132-30134, 2017. The CRISPR/Cas system includes a Cas protein andat least one or two ribonucleic acids that are capable of directing theCas protein to and hybridizing to a target motif in the CDK19 sequence.The Cas protein then cleaves the target motif and results in adouble-strand break or a single-strand break. Any CRISPR/Cas system thatis capable of altering a target polynucleotide sequence in a cell can beused in methods described here. In some embodiments, the CRISPR/Cassystem is a CRISPR type I system. In some embodiments, the CRISPR/Cassystem is a CRISPR type II system. In some embodiments, the CRISPR/Cassystem is a CRISPR type V system.

The Cas protein used in the methods described herein can be a naturallyoccurring Cas protein or a functional derivative thereof. A “functionalderivative” includes, but are not limited to, fragments of a nativesequence and derivatives of a native sequence polypeptide and itsfragments, provided that they have a biological activity in common withthe corresponding native sequence polypeptide. A biological activitycontemplated herein is the ability of the functional derivative tohydrolyze a DNA substrate (e.g., a CDK19 gene) into fragments. The term“derivative” encompasses both amino acid sequence variants ofpolypeptide, covalent modifications, and fusions thereof. Suitablederivatives of a Cas protein or a fragment thereof include but are notlimited to mutants, fusions, or covalent modifications of Cas protein.

In some embodiments, the Cas protein used in methods described herein isCas9 or a functional derivative thereof. In some embodiments, the Cas9protein is from Streptococcus pyogenes. Cas9 contains 2 endonucleasedomains, including an RuvC-like domain which cleaves target DNA that isnoncomplementary to crRNA, and an HNH nuclease domain which cleavestarget DNA complementary to crRNA. The double-stranded endonucleaseactivity of Cas9 also requires that a short conserved sequence (e.g.,2-5 nucleotides), known as a protospacer-associated motif (PAM), followsimmediately after the 3′ end of a target motif in the target sequence.

In some embodiments, the Cas protein is introduced into TNBC cells inpolypeptide form. In certain embodiments, the Cas protein may beconjugated to a cell-penetrating polypeptide. Non-limiting examples ofcell-penetrating peptides include, but are not limited to, e.g., thoseprovided in Milletti et al., Drug Discov. Today 17: 850-860, 2012, therelevant disclosure of which is hereby incorporated by reference in itsentirety. In other embodiments, a TNBC cell may be geneticallyengineered to produce the Cas protein.

In some embodiments, the target motif in the CDK19 gene, to which theCas protein is directed by the guide RNAs, may be between 15 and 25nucleotides in length (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25 nucleotides in length). In some embodiments, the target motif is atleast 20 nucleotides in length. In some embodiments, the target motif inthe CDK19 gene immediately precedes a short conserved sequence known asa protospacer-associated motif (PAM), recognized by the Cas protein. Insome embodiments, the PAM motif is an NGG motif. In some embodiments,the target motif of the CDK19 gene is within the first exon. In someembodiments, the target motifs can be selected to minimize off-targeteffects of the CRISPR/Cas systems. Those skilled in the art willappreciate that a variety of techniques can be used to select suitabletarget motifs for minimizing off-target effects (e.g., bioinformaticsanalyses).

The ribonucleic acids that are capable of directing the Cas protein toand hybridizing to a target motif in the CDK19 gene are referred to assingle guide RNA (“sgRNA”). The sgRNAs can be selected depending on theparticular CRISPR/Cas system employed, and the sequence of the targetpolynucleotide, as will be appreciated by those skilled in the art. Insome embodiments, the one or two ribonucleic acids can also be selectedto minimize hybridization with nucleic acid sequences other than thetarget polynucleotide sequence. In some embodiments, the one or tworibonucleic acids are designed to hybridize to a target motifimmediately adjacent to a deoxyribonucleic acid motif recognized by theCas protein. Guide RNAs can also be designed using available software,for example, CRISPR Design Tool (Massachusetts Institute of Technology).In some embodiments, the one or more sgRNAs can be transfected into TNBCcells, according to methods known in the art.

The use of antibodies for therapeutic purposes has been used to treatcancer. Passive immunotherapy involves the use of monoclonal antibodies(mAbs) in cancer treatments (see for example, Devita, Hellman, AndRosenberg’s Cancer: Principles & Practice Of Oncology, Eighth Edition(2008), DeVita, V. et al. Eds., Lippincott Williams & Wilkins,Philadelphia, Pa., pp. 537-547, 2979-2990). These antibodies can haveinherent therapeutic biological activity both by direct inhibition oftumor cell growth or survival and by their ability to recruit thenatural cell killing activity of the body’s immune system. Theantibodies can be administered alone or in conjunction with radiation orchemotherapeutic agents. Trastuzumab, approved for treatment of breastcancer is an example of such a therapeutic. Alternatively, antibodiescan be used to make antibody-drug conjugates in which the antibody islinked to a drug and directs that agent to the tumor by specificallybinding to the tumor. Ado-Trastuzumab emtansine (T-DM1) is an example ofan approved antibody-drug conjugate used for the treatment of breastcancer (see, Deng et al., Curr. Med. Chem., Vol. 24(23), 2505-2527(2017). Another type of immunotherapy is active immunotherapy, orvaccination, with an antigen present on a specific cancer (e.g., TNBCcells) or a DNA construct that directs the expression of the antigen,which then evokes the immune response in the subject, i.e., to inducethe subject to actively produce antibodies against their own cancer.

Antibodies have been highly effective in targeting cell surface proteinsinvolved in disease. Though it is generally believed that their largesize, complex architecture, and structural reliance on disulfide bondspreclude intracellular application, a number of examples of both insitu-expressed (see, e.g, Miersch and Sidhu, F1000Res doi:10.12688/f1000research.8915.1, 2016) and exogenously supplied wholeantibodies shown to maintain functional intracellular activity exist inthe literature (see, e.g., Biocca et al., Expression and targeting ofintracellular antibodies in mammalian cells. EMBO J. (1990); 9(1): 101-8and Steinberger et al., Functional deletion of the CCR5 receptor byintracellular immunization produces cells that are refractory toCCRS-dependent HIV-1 infection and cell fusion. Proc Natl Acad Sci USA.(2000); 97(2): 805-10). Attempts to use smaller, less complex bindingproteins such as antigen-binding fragments (Fabs) and single-chainvariable fragments (scFvs) for intracellular application have similarlyshown success in their ability to bind and modulate cytoplasmic proteinfunction (See for example, Marasco et al., Design, intracellularexpression, and activity of a human anti-human immunodeficiency virustype 1 gp120 single-chain antibody. Proc Natl Acad Sci USA. (1993);90(16): 7889-93).

As used herein, the term “antibody” encompasses, but is not limited to,whole immunoglobulin (i.e., an intact antibody) of any class. Nativeantibodies are usually heterotetrameric glycoproteins, composed of twoidentical light (L) chains and two identical heavy (H) chains.Typically, each light chain is linked to a heavy chain by one covalentdisulfide bond, while the number of disulfide linkages varies betweenthe heavy chains of different immunoglobulin isotypes. Each heavy andlight chain also has regularly spaced intrachain disulfide bridges. Eachheavy chain has at one end a variable domain (V(H)) followed by a numberof constant domains. Each light chain has a variable domain at one end(V(L)) and a constant domain at its other end; the constant domain ofthe light chain is aligned with the first constant domain of the heavychain, and the light chain variable domain is aligned with the variabledomain of the heavy chain. Particular amino acid residues are believedto form an interface between the light and heavy chain variable domains.The light chains of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (κ) andlambda (A), based on the amino acid sequences of their constant domains.Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively.

As used herein, the term “epitope” is meant to include any determinantcapable of specific interaction with the provided antibodies. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Identification of the epitope that theantibody recognizes is performed as follows. First, various partialstructures of the target molecule that the monoclonal antibodyrecognizes are prepared. The partial structures are prepared bypreparing partial peptides of the molecule. Such peptides are preparedby, for example, known oligopeptide synthesis technique or byincorporating DNA encoding the desired partial polypeptide in a suitableexpression plasmid. The expression plasmid is delivered to a suitablehost, such as E. coli, to produce the peptides. For example, a series ofpolypeptides having appropriately reduced lengths, working from the C-or N-terminus of the target molecule, can be prepared by establishedgenetic engineering techniques. By establishing which fragments reactwith the antibody, the epitope region is identified. The epitope is moreclosely identified by synthesizing a variety of smaller peptides ormutants of the peptides using established oligopeptide synthesistechniques. The smaller peptides are used, for example, in a competitiveinhibition assay to determine whether a specific peptide interferes withbinding of the antibody to the target molecule. If so, the peptide isthe epitope to which the antibody binds. Commercially available kits,such as the SPOTs Kit (Genosys Biotechnologies, Inc., The Woodlands, TX)and a series of multipin peptide synthesis kits based on the multipinsynthesis method (Chiron Corporation, Emeryvile, CA) may be used toobtain a large variety of oligopeptides.

The term antibody or fragments thereof can also encompass chimericantibodies and hybrid antibodies, with dual or multiple antigen orepitope specificities, and fragments, such as F(ab′)2, Fab′, Fab and thelike, including hybrid fragments. Thus, fragments of the antibodies thatretain the ability to bind their specific antigens are provided. Forexample, fragments of antibodies which maintain CDK19 binding activityare included within the meaning of the term antibody or fragmentthereof. Such antibodies and fragments can be made by techniques knownin the art and can be screened for specificity and activity according togeneral methods for producing antibodies and screening antibodies forspecificity and activity (See Harlow and Lane. Antibodies, A LaboratoryManual. Cold Spring Harbor Publications, New York (1988)).

Also included within the meaning of antibody or fragments thereof areconjugates of antibody fragments and antigen binding proteins (singlechain antibodies) as described, for example, in U.S. Pat. No. 4,704,692,the contents of which are hereby incorporated by reference in theirentirety.

In one embodiment, a therapeutic antibody (or antibody fragment) can beprepared using methods known in the art, having specificity for anantigen present in breast cancer, and in particular TNBC cells, that isabsent or present only at low levels in any normal (non-cancerous)tissue. The therapeutic antibody would therefore have biologicalactivity against TNBC cells and be able to recruit the immune system’sresponse to treat the disease. The therapeutic antibody can beadministered as a therapeutic alone or in combination with currenttreatments (such as chemotherapy, radiation, or platinum-basedtherapies) or used to prepare immunoconjugates linked to toxic agents,such as drugs.

Monoclonal antibodies to CDK19 (e.g., anti-CKD19 antibodies), made bymethods known in the art, can be used to identify the presence orabsence of cancerous cells in breast tissue, for purposes of diagnosisor treatment. Anti-CKD19 antibodies can also be used to identify thepresence or absence of cancerous cells, or the level thereof, which arecirculating in the blood after their release from a solid tumor. Suchcirculating antigen can include an intact CDK19 antigen, or a fragmentthereof that retains the ability to be detected according to the methodstaught herein. Such detection may be effected for example, by FACSanalysis using standard methods commonly used in the art.

In some embodiments, methods of targeting CDK19 can includeadministering to a subject in need thereof, a therapeutically effectiveamount of an antibody (e.g., an anti-CKD19 antibody) that isimmunoreactive to CDK19 for the treatment of breast cancer, inparticular treatment of TNBC. In one embodiment, the antibody havingimmunoreactivity to CDK19 targets intracellular signaling molecules,such as kinases, as opposed to cell surface molecules, whereby thespecificity of the antibody is provided by neutralizing epitope(s)present on CDK19 that are not present on CDK8. In another embodiment,the anti-CDK19 antibody can target the Pl3K/mTOR/AKT pathway or ERK5(see, Ocana and Pandiella, Oncotarget, 8(13), 22218-22234 (2017)). Inone embodiment, the anti-CDK19 antibody can target multipleintracellular signaling molecules, for example, the Pl3K/mTOR andJAK/STAT pathway. In yet another embodiment, the anti-CDK19 antibody cancomprise an engineered protein that binds to a neutralizing epitopepresent on CDK19 that is not present on CDK8.

In one embodiment, methods of targeting CDK19 can include administeringto a subject in need thereof, a therapeutically effective amount of atumor antigen (TA)-specific monoclonal antibody for the treatment ofTNBC. In one embodiment, the TA-specific mAB can be directed to anintracellular antigen associated with TNBC (See for example, Wang etal., Molecular Oncology, Vol. 9(10), (2015) 1982-1993 and Just, FEBSletters, 2:21 (2014), 350-355).

In one aspect, provided is a method of treating a subject with breastcancer, the method including the step of administering to the subject apharmaceutically effective amount of a composition comprising a CDK19targeting agent. The CDK19 targeting agent may be a CDK19 targetedantibody, a CDK19 targeted peptide, a CDK19 targeted small molecule, aCDK19 targeted RNA molecule, or a combination thereof. In someinstances, the CDK19 targeted agent may be conjugated to a therapeuticagent. In some instances, the method further includes administering asecond form of cancer therapy (e.g., chemotherapy or radiation therapy)to the subject. In one embodiment, the breast cancer is TNBC. In anotheraspect, provided is a method of inhibiting expression of the CDK19 genein a breast cancer cell, the method including the steps of contacting abreast cancer cell expressing the CDK19 gene with a synthetic CDK19targeted RNA molecule.

In another aspect, provided is a method of assessing responsiveness of asubject with cancer to a CDK19 targeted agent including the steps of:(a) measuring in a tumor sample from a subject the amount of CDK19; (b)determining if a subject has a cancer characterized as having a highlevel of CDK19 expression; and (c) indicating that the subject is morelikely to respond to the CDK19 targeted agent if the subject’s cancer ischaracterized as having a high level of CDK19 expression or that thesubject is less likely to respond to the CDK19 targeted agent if thesubject’s cancer is characterized as having a low level of CDK19expression.

In one aspect, provided is a method of treating a subject with cancer,the method comprising administering to the patient a pharmaceuticallyeffective amount of a composition comprising a CDK19 targeted agent. TheCDK19 targeted agent is an agent that specifically binds to CDK19protein or to CDK19 mRNA. CDK19 targeted agents include antibodies, orfragments thereof, peptides, small molecules, and polynucleotides (suchas RNA molecules) that specifically bind to CDK19 protein or to CDK19mRNA. The composition may further comprise a pharmaceutically acceptablecarrier. In some instances, CDK19 targeted agents that bind to the CDK19protein may directly inhibit CDK19 activity. In other instances, CDK19targeted agents that bind to CDK19 mRNA may inhibit CDK19 expression andthereby inhibit CDK19 activity.

In one instance, the CDK19 targeted agent may comprise a CDK19 targetedantibody. The CDK19 targeted antibody may be a monoclonal antibody. Insome instances, the CDK19 targeted antibody may be a humanized antibody.In another instance, the CDK19 targeted agent may be a CDK19 targetedpeptide. In yet another instance, the CDK19 targeted agent may be aCDK19 targeted small molecule. The CDK19 targeted peptides and smallmolecules may be derived in a variety of manners as discussed furtherbelow. In some instances, the peptides are derived from the sequence ofa CDK19 targeted antibody.

In some instances, treating a subject with the methods described hereininhibits at least one of: formation of a tumor, the proliferation oftumor cells, the growth of tumor cells, or metastasis of tumor cells inthe subject. In another embodiment, treating a subject with the methodsdescribed herein may result in reduction of tumor size and, in someinstances, elimination of one or more tumors in the subject.

3.1.4. Small Molecule Inhibitors

In one approach, methods for treating TNBC include targeting the CDK19protein using a small molecule inhibitor of CDK19 activity. Examples ofsmall molecule inhibitors of CDK19 are described in U.S. Pat. No.9,321,737, U.S. Pat. Publication No. US 20170071942, Mallinger et al.,J. Med. Chem. 59:1078, 2016, and Czodrowski et al., J. Med. Chem.59:9337, 2016. In some embodiments, the small molecule inhibitors bindto the ATP binding site of CDK19 to inhibit its activity.

The small molecule inhibitor of CDK19 may bind to the ATP binding siteof CDK19 covalently or non-covalently to inhibit its activity. In otherembodiments, the small molecule inhibitor may bind to other parts ofCDK19 outside of the ATP binding site. For example, the small moleculeinhibitor may form a covalent interaction with an amino acid (e.g.,methionine, tyrosine, or serine) outside of the ATP binding site toinhibit CDK19 activity. In addition to occupying the ATP binding toinhibit kinase activity, a small molecule inhibitor may also bind toCDK19 to cause a conformational change in CDK19 that prevents CDK19 fromfunctioning. In some embodiments, the small molecule inhibitor may bindto CDK19 with a higher affinity than to CDK8. As shown in FIG. 9 , thevast majority of amino acid differences between CDK19 and CDK8 are inthe C-terminal domain. In some embodiments, without being bound by anytheory, a small molecule inhibitor may bind to an amino acid or aportion in the C-terminal domain of CDK19, that is different from thecorresponding amino acid or portion of CDK8, to achieve selectiveinhibition of CDK19 over CDK8.

In some embodiments the small molecule inhibitor is other than acompound described in U.S. Pat. No. 9,321,737. In some embodiments thesmall molecule inhibitor is other than a compound described in U.S. Pat.Publication No. US 20170071942. In some embodiments the small moleculeinhibitor is other than a compound described in, Mallinger et al., J.Med. Chem. 59:1078, 2016. In some embodiments the small moleculeinhibitor is other than a compound described in Czodrowski et al., J.Med. Chem. 59:9337, 2016. In some embodiments the small moleculeinhibitor is other than one or more compounds selected from the groupconsisting of Cortistatin A, Sorafenib, Linifanib, Ponatinib, Senexin B,CCT251545, and CCT251921

3.1.5. Cdk19 Inhibitors That Do Not Significantly Inhibit Expression orActivity of Cdk8 or Which Inhibits Expression or Activity of Cdk19 to aGreater Extent Than It Inhibits Expression or Activity of CDK8

Agents that inhibitors expression or activity of CDK19 but do notinhibit expression or activity of CDK8, or agents that inhibitexpression or activity of CDK19 to a greater extent than expression oractivity of CDK8 is inhibited can be designed based on differences insequence and structure of the CDK19 and CDK8 proteins and theircorresponding genes and mRNAs. For example, an alignment of CDK19 andCDK8 mRNA sequences can identify non-identical or low identitynucleotide sequences that can be used to design shRNAs or other nucleicacid agents that associate with CDK19 mRNA but not CDK8 sequences. (see,FIGS. 16 and 17 ). Likewise, aligning CDK19 and CDK8 amino acidsequences can identify divergent regions and antibodies or other bindingagents can be produced to specifically bind the CDK19 protein. Likewise,small molecule agents can be identified (by rational drug design orscreening) that specifically inhibit CDK19 activity or inhibit CDK19activity to a greater degree that CDK8 activity.

The term “an agent that inhibits CDK19 activity but does notsignificantly inhibit activity of CDK8” as used herein, refers to anagent that is capable of specifically binding and inhibiting theactivity of CDK19 such that minimal CDK19 activity is detected in vivoor in vitro; while the agent causes no significant decrease in CDK8activity under the same conditions. For example, an agent that inhibitsactivity of CDK19 can specifically bind to CDK19 and fully orsignificantly inhibit CDK19 activity in vivo or in vitro. In some cases,a CDK19 inhibitor can be identified by its ability to preferentiallybind to the CDK19 gene or a CDK19 gene product, and fully inhibitexpression or activity of CDK19. Inhibition of CDK19 occurs when CDK19activity, when exposed to an agent, is at least about 70% less, forexample, at least about 75%, 80%, 90%, or 95% less than CDK19 activityin the presence of a control or in the absence of the agent. Nosignificant decrease in CDK8 activity occurs when CDK8 activity, uponexposure to the agent, is at least about 90%, for example, at least 95%,96%, 97%, 98%, 99%, or 100%, in comparison to CDK8 activity in theabsence of the agent. As set forth herein, the agent can include smallmolecules (i.e., a molecule having a formula weight of 1000 Daltons orless), such as small molecule chemical inhibitors or large molecules,such as siRNA, shRNA, antisense oligonucleotides, or proteins.

Determining the effect of the agent on CDK19 and/or CDK8 activity can bemeasured using one or more methods known in the art, including but notlimited to, half maximal inhibitory concentration (IC₅₀), dissociationconstant (K_(D)), and inhibitor constant (K_(l)). For example, IC₅₀ is ameasure of the effectiveness of a substance in inhibiting a specificbiological or biochemical function. This value indicates theconcentration of the substance needed to inhibit a given biologicalprocess (or component of the biological process) by half. The IC₅₀values are typically expressed as molar concentration. According to theFood and Drug Administration (FDA), IC₅₀ represents the concentration ofa drug required for 50% inhibition in vitro. In one embodiment, an agentthat inhibits CDK19 activity but does not significantly inhibit activityof CDK8 has an IC₅₀ that is at least about 2-fold, 5-fold, 10- fold,50-fold, 75-fold, or 100-fold, lower than the concentration of the agentrequired to effect CDK8 activity under the same conditions. In anotherembodiment, the IC₅₀ for the agent to inhibit CDK19 activity is at leastabout 25%, 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, lowerthan the IC₅₀ for the agent to inhibit the activity of CDK8.

In another embodiment, the effect of the agent on CDK19 and CDK8activity can be determined by calculating the equilibrium dissociationconstant (K_(D)) of the agent to each CDK. For example, an agent thatinhibits the activity of CDK19 but does not significantly inhibitactivity of CDK8 has a K_(D) that is at least about 2-fold, 5-fold, 10-fold, 50-fold, or 100-fold lower than the K_(D) of the agent to CDK8under the same conditions. In one embodiment, the K_(D) for the agent(to CDK19) is at least about 25%, 50%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99%, lower than the Ko for the agent (to CDK8). In apreferred embodiment, the K_(D) is lower for the agent to CDK19 ascompared to the K_(D) of the agent to CDK8. Said differently, theequilibrium dissociation constant of the agent (to CDK8) is greater thanthe equilibrium dissociation constant of the agent (to CDK19). In oneembodiment, the agent can include an antibody having a K_(D) value inthe micromolar (10⁻⁶) to nanomolar (10⁻⁷ to 10⁻⁹) range. In anotherembodiment, the agent can include an antibody having a K_(D) in thenanomolar range (10⁻⁹) to the picomolar (10⁻¹²) range. In yet anotherembodiment, the agent can have a nanomolar (nM) equilibrium dissociationconstant to CDK19 and a micromolar (µM) equilibrium dissociationconstant to CDK8. U.S. Pat. Publication No. US20120071477 describeskinase inhibition assays in which a compound at a single concentration(2,000 nM) to inhibit ATP pocket binding.

In another embodiment, the effect of the agent on CDK19 and CDK8activity can be determined by calculating the inhibitor constant (K_(l))of the agent to each CDK. The K_(l) is an indication of how potent aninhibitor is; it is the concentration required to produce half maximuminhibition. The lower the Ki, the greater the binding affinity betweenthe agent and the CDK gene. For example, an agent that inhibits theactivity of CDK19 but does not significantly inhibit activity of CDK8has a K_(l) that is at least about 2-fold, 5-fold, 10- fold, 50-fold,75-fold, or 100-fold lower than the K_(l) of the agent (to CDK8) underthe same conditions. In one embodiment, the K_(l) for the agent to CDK19is at least about 25%, 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99%, lower than the K_(l) for the agent to CDK8. In a preferredembodiment, the K_(l) is lower for the agent to CDK19 as compared to theK_(l) of the agent to CDK8. Said differently, the inhibitor constant ofthe agent to CDK8 is greater than the inhibitor constant of the agent toCDK19. For example, an agent that inhibits activity of CDK19 can bind toCDK19 and significantly inhibit CDK19 activity in vivo or in vitro. Insome cases, a CDK19 inhibitor can be identified by its ability topreferentially bind to CDK19 and fully inhibit activity of CDK19.Inhibition of CDK19 occurs when CDK19 activity, when exposed to an agentof the invention, is at least about 70% less, for example, at leastabout 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% less, or totally inhibited,in comparison to CDK19 activity in the presence of a control or in theabsence of the agent. No significant decrease in CDK8 activity occurswhen, CDK8 activity upon exposure to the agent, is at least about 90%,for example, at least 95%, 96%, 97%, 98%, 99%, or 100%, in comparison toCDK8 activity in the absence of the agent.

The term “an agent that inhibits activity of CDK19 to a greater extentthan it inhibits activity of CDK8” as used herein, refers to an agentthat is capable of binding and inhibiting the activity of CDK19significantly more than the agent’s effect on inhibiting the activity ofCDK8 under the same conditions. For example, an agent that inhibitsactivity of CDK19 to a greater extent than inhibiting the activity ofCDK8, occurs when CDK19 activity, when exposed to an agent of theinvention, is at least about 10% less, for example, at least about 15%,20%, 30%, 40%, or 50% less, than the activity of CDK8 under the sameconditions in vitro or in vivo. In a preferred embodiment, an agentinhibits the activity of CDK19 to a greater extent than the activity ofCDK8, when the activity of CDK19 observed is at least 10% less than theactivity of CDK8 under the same conditions. In another embodiment, anagent inhibits the activity of CDK19 to a greater extent than CDK8activity, when at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or100-fold less CDK19 activity is observed as compared to CDK8 activityunder the same conditions. The extent of inhibition (i.e., comparingCDK19 activity to CDK8 activity) can be determined using one or moremethods known in the art, including but not limited to assays describedherein in the Examples section of the specification and for example,“Percent Of Control (POC)” or “Normalized Percent Inhibition (NPI)”. POCand NPI are methods that normalize data and are often used whencomparing multiple agents (e.g., various antibodies or small molecules)against multiple targets (e.g., CDK19 and CDK8). For example, POC is amethod that corrects for plate-to-plate variability (for example inhigh-throughput drug screening) by normalizing an agent’s measurementrelative to one or more controls present in the plate. Raw measurementsfor each agent can be divided by the “average” of within-plate controls.NPI is a control-based method in which the difference between the agentmeasurement and the mean of the positive controls is divided by thedifference between the means of the measurements on the positive and thenegative controls (Malo et al., Nature Biotechnology, Vol. 24, 167-175(2006)). By normalizing the extent of inhibition observed, accurateconclusions can be made regarding which agent(s) are effective atinhibiting the activity of each target under investigation.

3.1.6. Combination Therapy

In one approach the patient is treated with a combination therapycomprising an agent that inhibits expression or activity of CDK19 and(a) radiation therapy and/or chemotherapy. In one approach radiation orchemotherapy eliminates the bulk of the tumor mass and the CDK19inhibitor reduces the number of viable cancer stem cells (e.g.,EpCAM^(med/high)/CD10^(-/low)) cells. In one approach the chemotherapycomprises administration of an anthracycline (e.g., Doxorubicin orEpirubicin), a taxane (e.g., Paclitaxel or Docetaxel), ananti-metabolite (e.g., Capecitabine or Gemcitabine), a platinum agent(e.g., Carboplatin or Cisplatin), Vinorelbine, or Eribulin.

3.2 Methods of Assessing or Predicting Therapeutic Effect

A course of therapy with the CDK19 inhibitor will have a beneficialoutcome for the patient, including, for example, a reduction in tumorvolume, a reduction in metastases, and a reduction in tumor cells havingthe phenotype EpCAM^(med/high) and CD10^(-/low) _(.)

Tumor volume may be measured using art-known methods. See, e.g., Wapniret al., Breast Cancer Res Treat 41:15-19, 1996; Sapi et al., PLoS One10:e0142190, 2015. Tumor volume may be reduced by at least 10%,optionally at least 20% and sometimes by at least 50% after a course oftreatment with a CDK19 inhibiting agent as monotherapy or in combinationwith other agent(s) or treatments. In some embodiments, the reduction intumor volume (e.g., at least 10%, 20%, or 30% reduction in tumor volume)may be observed as soon as within 1 month of initiating therapy. Inother embodiments, the reduction in tumor volume (e.g., at least 10%,20%, 30%, 40%, 50%, or 60% reduction in tumor volume) may be observedwithin 2, 3, 4, 5, or 6 months of initiating therapy. In otherembodiments, the methods described herein to treat TNBC may also slowdown or inhibit the further growth of a tumor. In some embodiments apatient receives combination therapy and a therapeutic benefit isobserved that exceeds that of monotherapy with the second agent.

A reduction in metastases in an individual may be determined asdescribed in Makela et al., Sci Rep. 7:42109, 2017 and may be observedin a population according to standard methodology.

In some embodiments, the presence or amount of cancer cells having theexpression profile EpCAM^(med/high) and CD10^(-/low) in a TNBC tumortissue obtained from a subject may be used to predict or assess thetherapeutic responsiveness of the subject to treatments that target theCDK19 gene or its corresponding protein. As described and demonstratedherein, cells having the expression profileEpCAM^(med/high)/CD10^(-/low) have a high tumor initiating capacity andare also enriched in CDK19. In some embodiments, subjects having a highpercentage of EpCAM^(med/high) and CD10^(-/low) TNBC cells may beespecially responsive.

In one approach the likely therapeutic responsiveness of a subject withTNBC to a CDK19 targeting agent is determined by (a) quantitating EpCAM^(med/high) /CD10^(-/low) cells in a tumor sample obtained from thesubject; (b) comparing the quantity of EpCAM^(med/high)/CD10⁻ ^(/low)cells in (a) to a reference value characteristic of tumors responsive toa CDK19 targeting therapy, and treating the patient with an inhibitor ofCDK19 expression or activity if the quantity ofEpCAM^(med/high)/CD10^(-/low) cells is equal to or exceeds the referencevalue. The reference value can be determined by quantitatingEpCAM^(med/high)/CD10^(-/low) cells in healthy and TNBC populations andcalculating statistically significant ranges characteristic of healthyand tumor tissues. In another approach tumor tissue and healthy tissuefrom the same subject can be tested, and subjects with elevatedEpCAM^(med/high)/CD10^(-low) cells in tumor relative to healthy tissuescan be identified as likely to respond to CDK19 targeted therapy.

3.3 Delivery of Agents

The pharmaceutical compositions used in methods described herein mayinclude an active ingredient and one or more pharmaceutically acceptablecarriers or excipients, which can be formulated by methods known tothose skilled in the art. In some embodiments, a pharmaceuticalcomposition of the present invention includes, in a therapeuticallyeffective amount, a DNA or RNA oligonucleotide that decreases theexpression level of the CDK19 gene. In other embodiments, apharmaceutical composition of the present invention includes, apharmaceutical composition of the present invention includes a DNA orRNA oligonucleotide in a therapeutically effective amount, a smallmolecule that inhibits the activity of CDK19. The therapeuticallyeffective amount of the active ingredient in a pharmaceuticalcomposition is sufficient to prevent, alleviate, or ameliorate symptomsof a disease or to prolong the survival of the subject being treated.Determination of a therapeutically effective amount is within thecapability of those skilled in the art.

In certain embodiments, a pharmaceutical composition of the presentinvention is formulated as a depot preparation. In general, depotpreparations are typically longer acting than non-depot preparations. Insome embodiments, such preparations are administered by implantation(for example subcutaneously) or by intramuscular injection. In someembodiments, depot preparations are prepared using suitable polymeric orhydrophobic materials (for example an emulsion in an acceptable oil) orion exchange resins, or as sparingly soluble derivatives, for example,as a sparingly soluble salt.

In some embodiments, a pharmaceutical composition may include a deliverysystem. Examples of delivery systems include, but are not limited to,exosomes, liposomes, and emulsions. In some embodiments, an activeingredient may be loaded or packaged in exosomes that specificallytarget a cell type, tissue, or organ to be treated. Exosomes are smallmembrane-bound vesicles of endocytic origin that are released into theextracellular environment following fusion of mutivesicular bodies withthe plasma membrane. Exosome production has been described for manyimmune cells including B cells, T cells, and dendritic cells. Techniquesused to load a therapeutic compound into exosomes are known in the artand described in, e.g., U.S. Pat. Publication Nos. US 20130053426 and US20140348904, and International Patent Publication No. WO 2015002956,which are incorporated herein by reference. In some embodiments,therapeutic compounds may be loaded into exosomes by electroporation orthe use of a transfection reagent (i.e., cationic liposomes). In someembodiments, an exosome-producing cell can be engineered to produce theexosome and load it with the therapeutic compound. For example, exosomesmay be loaded by transforming or transfecting an exosome-producing hostcell with a genetic construct that expresses the active ingredient(i.e., a DNA or RNA oligonucleotide), such that the active ingredient istaken up into the exosomes as the exosomes are produced by the hostcell. Various targeting moieties may be introduced into exosomes, sothat the exosomes can be targeted to a selected cell type, tissue, ororgan. Targeting moieties may bind to cell-surface receptors or othercell-surface proteins or peptides that are specific to the targeted celltype, tissue, or organ. In some embodiments, exosomes have a targetingmoiety expressed on their surface. In some embodiments, the targetingmoiety expressed on the surface of exosomes is fused to an exosomaltransmembrane protein. Techniques of introducing targeting moieties toexosomes are known in the art and described in, e.g., U.S. Pat.Publication Nos. US 20130053426 and US 20140348904, and InternationalPatent Publication No. WO 2015002956, which are incorporated herein byreference.

4. Examples 4.1 Example 1- Materials and Experimental Methods ChemicalReagents

The following are the chemical names for the compounds used in thisstudy. CCT152921 is4-[(2-Phenylethyl)amino]-6-quinazolinecarbonitrile(NIH NCAT). The compound was re-suspended in vehicle (PBS + 0.5%Methocel (w/v) + 0.25% Tween 20 (v/v)) to a concentration of 3 mg/mL andmice were dosed at 30 mg/kg. CCT251921 or vehicle was administered viadaily oral gavage.

shRNA Expression Lentiviral Plasmids

Pairs of complementary ssDNA oligonucleotides containing the sensetarget sequence, a 15-mer loop sequence (5′-GTTAATATTCATAGC-3′ SEQ IDNO: 19), and the reverse complement of the sense sequence weresynthesized (Elim Biopharmaceuticals). The oligonucleotides wereannealed in 50 µM annealing buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl, 1mM EDTA). The double-stranded DNA oligo templates were subsequentlycloned into the pRSI12-U6-(sh)-HTS4-UbiC-TagRFP-2A-Puro shRNA expressionvector (Cellecta) digested with Bbsl for constitutively active shRNAvector constructs and pRSITUR-U6Tet-(sh)-UbiC-TetRep-2A-TagRFP digestedwith Bbsl for inducible shRNA vector constructs. The sense strands inthe shRNA vectors used in this study were: 5′-GCG AGA ATT GAA GTA CCTTAA-3′ (shCDK19-1 (SEQ ID NO: 1)), 5′-ACC AGC AAA TAT CCT AGT AAT-3′(shCDK19-2 (SEQ ID NO:2)), and 5′-GCA GGG TAATAA CCA CATTAA-3′ (shCDK8-2(SEQ ID NO: 3)). The unmodified pRSI12-U6-(sh)-HTS4-UbiC-TagRFP-2A-PuroshRNA expression vector above was used as the ‘empty’ control shRNA. ThepHIV-ZsGreen expression vector (Addgene) was used to produce GFPpositive tumor cells. The DECIPHER 27 K Pooled shRNA lentiviruslibrary - Human Module 1 (Cellecta) used for the RNAi screen contains27,500 unique shRNA constructs targeting 5,043 human genes(approximately five or six redundant shRNAs per gene) in the same pRSI12shRNA expression vector.

Cell Lines

MDA-MB231, MDA-MB468, HS578T, and 293T cells were obtained from ATCC.HMEC cells were obtained from ThermoFisher Scientific. These cells werecertified by the vendors to be mycoplasma free. None of the cell linesused are listed in the database of commonly misidentified cell linesmaintained by ICLAC. All cell lines used were passaged less than 10times from when the original cells from the vendors were thawed. AllMDA-MB231, MDA-MB468, 293T, and HS578T cells were grown in DMEM(Invitrogen) supplemented with PSA (Life Technologies), 10% FBS(Hyclone), Glutamax (ThermoFisher Scientific), and sodium pyruvate (LifeTechnologies). HMEC cells were grown in HuMEC Ready Medium (ThermoFisherScientific).

Mice

Nod scid gamma (NSG) mice (NOD.Cg-Prkdc^(scid) IL2Rg^(tm1Wjl)/SzJ) werepurchased from the Jackson Laboratory. Mice used for PDX experimentswere adult female mice between 8 and 10 weeks old. All the mice used inthis study were maintained at the Stanford Animal Facility in accordancewith a protocol approved by the Stanford University APLAC committee.Mice were maintained in-house under aseptic sterile conditions. Micewere administered autoclaved food and water. For PDX experimentsutilizing doxycycline inducible constructs, mice were provided rodentfeed containing 625 mg Doxycycline hyclate/kg diet (Envigo) in place oftheir normal rodent diet.

PDX Tumors and Their Pathological and Clinical Characteristics

For human samples, informed consent was obtained after the approval ofprotocols by the Institutional Review Boards of Stanford University andThe City of Hope. See FIG. 15 for a full description of all the PDXtumors used in this study.

Single Cell Suspension of PDX Tumor Cells

Xenografts were mechanically chopped with a razor blade to approximately1 mm pieces and then incubated at 37°-C for 3 to 4 hours withcollagenase and hyaluronidase (Stem Cell Technologies) in AdvancedDMEM/F12 (Invitrogen) with 120 µg/mL penicillin, 100 µg/mL streptomycin,0.25 µg/mL amphotericin-B (PSA) (Life Technologies). Cells were thentreated with ACK lysis buffer (Gibco) to lyse red blood cells, followedby 5 mins of treatment with pre-warmed dispase (Stem Cell Technologies)plus DNAsel (Sigma) and filtered through a 40 µm nylon mesh filter.Cells were finally washed with flow cytometry buffer (HBBS, 2% FCS,PSA).

Enrichment of PDX Tumor Cells

After PDX tumors were dissociated into single cells, the number of livecells were determined with Trypan blue staining and manually countedwith a hemocytometer. Cells were resuspended with flow cytometry bufferto a concentration of 10⁶ live cells/mL and incubated 1:50 (v/v) withBiotin anti-human CD326 (EpCAM) antibody (Biolegend) for 20 mins at4°-C. Cells were washed with flow cytometry buffer and then resuspendedto 80 µL and incubated with 20 µL anti-biotin microbeads (MiltenyiBiotec) for 20 mins at 4°-C. Cells were then washed with flow cytometrybuffer and resuspended in 500 µL of buffer. Cells were applied tomagnetized LS columns (Miltenyi Biotec), washed, and eluted off magnetper manufacturer’s protocol.

Lentivirus Production

Lentivirus was produced with Packaging Plasmid Mix (Cellecta) andsubcloned pRSl12 shRNA expression plasmids using Lipofectamine 2000(Thermofisher Scientific) in 293T cells per manufacturer’s instructions.Supernatants were collected at 48 h and 72 h, filtered with a 0.45 µmfilter and precipitated with Lentivirus Precipitation Solution (AlstemLLC) per manufacturer’s instructions. Virus was resuspended in 1/100original volume. Viral titers were determined by flow cytometry analysesof 293T cells infected with serial dilutions of concentrated virus.

Lentivirus Infection

For in vitro cell line experiments, concentrated lentiviral supernatant(to achieve an MOI of 3) was mixed with cells at the time of seeding.Cells were monitored by visualization of RFP under fluorescencemicroscopy. All flow cytometry analyses were performed after at least 72hours of infection.

For in vivo PDX tumor growth and organoid colony formation experiments,concentrated lentiviral supernatant (to achieve an MOI of 10) was mixedwith single cell suspensions of PDX tumor cells in organoid media with 4µg/mL of Polybrene (Sigma-Aldrich). Organoid media consisted of:Advanced DMEM/F12 (Invitrogen), 10% FBS (Hyclone), 2.5% growthfactor-reduced Matrigel (BD), 10 ng/mL mouse EGF (R&D), 100 ng/mL Noggin(R&D), 250 ng/mL RSPO-I (R&D), 1X B27 (Invitrogen), 1X N2 (Invitrogen),and PSA (Life Technologies). Cells were then spinoculated bycentrifuging at 15 ºC for 2 hours at 1200xg. Cells were resuspended bypipetting and left overnight in 48-well ultra-low attachment cellculture plates (Corning).

For organoid colony formation assays, cells were transferred the nextday to matrigel. For in vivo PDX assays, approximately 75% of the cellswere injected into NSG mice as described in the PDX tumor engraftmentsection. The remainder 25% of cells were plated on matrigel and grown inorganoid media for 72 hours until the cells became RFP positive. At thatpoint media was removed and exchanged for dispase and incubated for 2-3h until the matrigel dissolved. Dissociated cells were resuspended inflow cytometry buffer and analyzed by flow cytometry to determine the‘baseline’ RFP percentage for cells that were injected into the mice.

Organoid Colony Formation Assay

Irradiated L1-Wnt3a feeder cells (generous gift of Dr. Roel Nusse) weremixed with growth factor reduced matrigel (BD Biosciences) and allowedto solidify at 37 ºC. Single cell suspensions of PDX tumor cells weretransferred onto the solidified matrigel/feeder cell mix substrate andgrown in organoid media. Cells were grown for approximately 2 weeks in a37 ºC incubator with 5% CO₂. 50% of media was exchanged with fresh mediaevery 3-4 days. Colonies were counted under fluorescence microscopy.Only RFP positive colonies (which represent transduced cells) werecounted. For experiments in which we induced expression of CDK19 shRNA,doxycycline hyclate was added to a final concentration of 100 ng/mL intothe media.

Cell Viability Assay

For cell lines treated with chemical or infected with lentivirus, WST-1Cell Proliferation Reagent (Roche) was added at 1:10 (v/v) finaldilution to each well per manufacturer’s instructions. Cells weresubsequently incubated at 37 ºC and 5% CO₂. Between 1 and 4 hours afteraddition of reagent, plates were analyzed on a SpectraMax M3 Bioanalyzer(Molecular Devices). Absorbance for each well was measured at 450 nm(signal wavelength) and 650 nm (reference wavelength). Thus, the signalfor each experimental sample was Absorbance_(experimental)(A_(450nm)-A_(650nm)). To correct for the effect of media,Absorbance_(background) (A_(450nm)-A_(650nm)) was obtained by measuringabsorbance in a blank well. Thus, the background corrected signal foreach sample A_(corrected) = Absorbance_(experimental)-Absorbance_(background). All A_(corrected) values for the knockdownswere normalized to the A_(corrected) value for the control sample toobtain a ‘Relative Viability’.

Quantitative PCR RNA Expression Analyses

Cells were lysed with Trizol (Life Technologies) and RNA was extractedaccording to the manufacturer’s instruction. RNA was then treated withDNAsel to remove contaminating genomic DNA. RNA was reverse transcribedto cDNA using SuperScript III First Strand Synthesis kit (LifeTechnologies) according to the manufacturer’s instructions. TaqMan GeneExpression Master Mix (Applied Biosystems) and the following TaqMan GeneExpression Assays (Applied Biosystems) were used followingmanufacturer’s instructions: ACTB, Hs00357333_g1; CDK19, Hs01039931_m1;CDK8, Hs00993274_m1. Data was collected on a 7900HT Fast Real-Time PCRSystem (Applied Biosystems) and data analyzed with SDS 2.4 software(Applied Biosystems). Gene expression data in each sample was normalizedagainst the expression of beta-actin.

PDX Tumor Cell Engraftment and Limiting Dilution Assays

Single cell suspensions of PDX cells were resuspended in 50% (v/v)mixtures of normal matrigel (BD Biosciences) and flow cytometry bufferin a total volume of 50-100 µL. Using an insulin syringe, cells wereinjected subcutaneously into the nipple of female NSG mice at the fourthabdominal fat pad. For limiting dilution assays, the specific number ofcells injected into the mice were determined by flow cytometry andsecondarily by manual counting with a hemocytometer.

PDX Tumor Growth and Total Body Weights

PDX tumors were detected by palpation. Tumor volumes were determined bymeasuring the length (l) and width (w) and calculating volumes using theellipsoid formula ⅙ x l x w² x π. Tumors volumes and mice weights weredetermined twice per week.

Mouse PDX Tumor and Lung Dissection

Xenograft tumors and mice lungs were surgically resected after the micewere euthanized. A 3 to 4 mm section is cut from each tumor and saved inice cold PBS for imaging. The mice lungs and tumors were imaged on aM205FA Fluorescence Stereo Microscope (Leica) and images were capturedwith a DFC310FX camera (Leica).

Flow Cytometry to Determine RFP Percentage

Flow cytometry was performed with a 100 µm nozzle on a Flow CytometryAria II (BD Biosciences) with Diva software (BD Biosciences). Dataanalysis was performed using Flowjo software (Flowjo). For allexperiments, side scatter and forward scatter profiles (area and width)were used to eliminate debris and cell doublets. Dead cells wereeliminated by excluding 4′,6-diamidino-2-phenylindole (DAPI)-positivecells (Molecular Probes). For PDX tumor cells, they were gated for GFPpositivity and then for RFP positivity. RFP percentage is the percentageof GFP positive cells that are also RFP positive. For each sample, weobtain the RFP fraction that is: the RFP % in the tumor divided by thebaseline RFP % (see ‘Lentivirus infection’ section). RFP fraction foreach sample is then normalized to the RFP fraction for the shRNA controlsample which is set at 100% to obtain the ‘Normalized % RFP’.

Flow Cytometry Using EpCAM, CD10, and CD49f Cell Surface Markers forAnalysis and Cell Sorting

Flow cytometry for analysis and cell sorting was performed as previouslydescribed. Human antibodies used included: EpCAM-Alexa Fluor 488 (clone9C4, Biolegend); 1 µg mL⁻¹, CD49f-APC (clone GoH3, Biolegend); CD10PeCy7/Apc-Cy7 (clone H110a, Biolegend); 1 µg mL⁻¹ and H-2Kdbiotin/Pacific Blue (clone SF1-1.1, Biolegend); 1 µg mL⁻¹.

RNAi Dropout Viability Screen

GFP positive PDX-T1 tumors grown in NSG mice were dissected, processedto single cells, and enriched with EpCAM as described previously.Analysis of cells at this point showed that they were approximately98%-100% GFP positive.

For the in vitro RNAi dropout viability screen, 60 million dissociatedPDX-T1 cells were transduced with the DECIPHER 27 K Pooled shRNAlentivirus library-Human Module 1 (Cellecta) at an MOI of 1 in thepresence of polybrene and then spinoculated for 2 hours as describedpreviously. The next day, half the cells were spun down and frozen asthe in vitro baseline reference sample. A small number of cells wereplated separately in organoid colony formation conditions to determinelentiviral infection percentage after 72 hours (cells were found to beapproximately 80% RFP positive). The remainder of the cells were platedinto twelve 150 mm dishes prepared with 12 mL matrigel containingirradiated L1-Wnt3a feeder cells at 250,000 cells/mL of matrigel. Thecells were grown for 19 days with an exchange for fresh media every 3-4days. On the final day, all the media was exchanged with dispase inorder to dissolve the matrigel and to recover the cells. The cells fromall the plates were pooled, washed, and frozen as the in vitro organoidgrowth experimental sample.

For the in vivo RNAi dropout viability screen, 30 million dissociatedPDX-T1 cells were transduced with the DECIPHER 27 K Pooled shRNAlentivirus library-Human Module 1 (Cellecta) at an MOI of 1.25 in thepresence of polybrene and then spinoculated for 2 hours as describedpreviously. The next day, half the cells were spun down and frozen asthe in vivo baseline reference sample. A small number of cells wereplated separately in organoid colony formation conditions to determinelentiviral infection percentage after 72 hours (cells were found to beapproximately 70% RFP positive). The remainder of the cells wereresuspended in 50% (v/v) mixtures of normal matrigel (BD Biosciences)and flow cytometry buffer in a total volume of 1.8 mL. These cells wereinjected evenly into the right and left mammary fat pads of seventeenNSG mice. When tumors reached approximately 10 mm in diameter, the micewere euthanized and the tumors dissected as previously described. Thesetumors were then processed into single cells, pooled, washed, and frozenas the in vivo growth experimental sample.

The two pairs of samples, in vitro baseline reference sample and invitro organoid growth experimental sample and in vivo baseline referencesample and in vivo growth experimental sample, were submitted toCellecta, Inc. for genomic DNA extraction, bar code amplification,high-throughput sequencing and de-convolution. Twenty million barcodereads were performed for each sample.

‘Hit’ Selection Algorithm From the In Vivo and In Vitro RNAi DropoutViability Screens

Please see the schematic in FIG. 5C for an overview. We applied analgorithm to narrow our hits to a more manageable number forvalidation. 1) for each individual shRNA we determined a ‘dropout ratio’that was shRNA barcode counts in the growth experimental sample dividedby shRNA barcode counts in the baseline reference sample. In eachscreen, these were ranked from lowest to highest. 2) We examined the top5% of the lowest dropout ratios in each experiment and identified genestargeted by ≥ 2 shRNA. 3) We cross-referenced the shRNA gene targets inthe in vivo screen (208 genes) with those in the in vitro screen (150genes) to identify genes that overlapped between the two experiments.These 46 overlapping ‘hit’ genes are shown in FIG. 5A.

Immunofluorescence of PDX Tumors

Sections of the PDX tumors were fixed in formalin overnight and thentransferred to 70% ethanol. Samples were then embedded in paraffin andsectioned for histology. Formalin fixed paraffin embedded sections werede-parafinized in xylene and rehydrated in an ethanol gradient. Antigenretrieval was performed in a Tris-EDTA buffer by heating in a microwavefor 20 min. The primary antibodies, polyclonal Rabbit anti-CDK19 (Sigma)and polyclonal chicken anti-CDK8 (Novus Biologicals), were diluted 1:50and 1:100, respectively, in TBS + 1% BSA before applying to samplesovernight. After overnight incubation, the secondary antibodies, Cy3Donkey anti-Rabbit (Jackson ImmunoResearch) and Alexa 488 Goatanti-Chicken (Life Technologies) were diluted 1:500 in TBS + 1% BSA andincubated with the samples at room temperature. After DAPI staining,sections were mounted with Prolong® Gold antifade (Cell Signaling). AZeiss LSM710 Confocal microscope (Carl Zeiss) was used to take theimmunofluorescence images. Images for publication were processed withFiji software.

Microarray Experiment

EpCAM enriched PDX-T1 cells were infected with shCDK19-2, shCDK8-2 orcontrol shRNA and grown in organoid culture conditions for 72 hours.They were subsequently recovered from matrigel with dispase, resuspendedin flow cytometry buffer and sorted by flow cytometry to obtain cellsthat were both GFP and RFP positive. RNA was extracted from these cellsby RNeasy plus micro kit (Qiagen) according to manufacturer’sinstructions and quantified on an Agilent 2100 Bioanalyzer. 50 ng oftotal RNA from each sample was used. In vitro transcription,fragmentation, labeling, hybridization to the microarray and scanningwas performed by the Stanford Protein and Nucleic acid facility (PANfacility). Samples were hybridized on PrimeView Human Gene ExpressionArrays (Affymetrix). Gene Level Differential Expression Analysis wasperformed with the Transcriptome Analysis Console (Affymetrix).Downregulated genes were defined as those for which log₂(sample/control) < -1.5 and upregulated genes log₂ (sample/control) >1.5.

H3K27Ac Chromatin Immunoprecipitations

ChIP assays were performed as described in, e.g., Zarnegar et al.,Nucleic Acids Research, gkx648, July, 2017. Approximately 250,000 to500,000 MDA-MB231 cells were used per ChIP. 1 µg of anti-H3K27ac (ActiveMotif #39133) were used per ChIP.

Library Construction

ChIP enriched DNA was quantified using a Qubit 3.0 and dsDNA HS assay.Up to 1 ng of DNA was used for library construction using transpositionbased NEXTERA XT (followed manufacturer’s protocol with ~14 PCR cyclesfor indexing). Indexed samples were pooled and submitted for sequencingon a NextSeq500 to obtain 75 bp single end reads with read depths of ~60million reads.

Sequence Analysis

Raw sequence reads were uploaded to Galaxy (usegalaxy.org) and alignedto the human genome (hg19) using Bowtie2 (-very-fast-local). Onlyuniquely mapped reads were retained for further analysis. To visualizedata, alignment files were used to produce signal tracks with DeepTools(100 bp bins with 200 bp read extensions and RPKM normalization) andBigWig files were loaded into Broad’s Integrated Genome Browser. MACS2was used to call peaks (-nomodel, p=0.01, -broad, cuttoff 0.1,duplicates = auto, extension 200) for each replicate. A consensus peaklist containing only those peaks occurring in all replicates, wasgenerated using Bedtools. We performed differential peak analysis acrossconsensus peaks using DiffBind. The DiffBind output peak list wasannotated by fetching the nearest nonoverlapping feature of the humanRefSeq table from UCSC. Data for aggregation plots of ChIP signal acrossvarious peaks sets were generated using DeepTools′ computeMatrix(scale-regions: 1000; 50 bp bins) and plotProfile. Data was then plottedwith GraphPad Prism software.

GSEA Analysis

Gene set enrichment analysis (GSEA) was performed using the javaGSEAdesktop application (GSEA 3.0) with log₂ fold change values for CDK19knockdown versus Control as the ranking metric and Hallmarks,CDK19KD-EnhancerUp and CDK19KD-EnhancerDOWN as the gene sets that weretested for enrichment.

Metascape Analysis

Metascape custom enrichment analysis of Hallmark gene sets using theCDK19KD-EnhancerUP ‘core’ genes and the CDK19KD-EnhancerDOWN ‘core’genes (using the following parameters: H. Sapiens as the input species,p-value cutoffs of 0.01 and minimum enrichment 1.5) was performed online(www.metascape.org).

Statistical Analysis

Results are shown as mean ± s.d. Statistical calculations were performedwith GraphPad Prism software (GraphPad Software Inc). Variance wasanalyzed using the F-test. To determine P-values, t-test was performedon homoscedastic populations, and t-test with Welch correction wasapplied on samples with different variances. For animal studies, samplesize was not predetermined to ensure adequate power to detect apre-specified effect size, no animals were excluded from analyses,experiments were not randomized and investigators were not blinded togroup allocation during experiments.

4.2 Example 2 - Identification of Genes Essential for TNBC Growth

To identify genes essential for the growth of TNBC, two pooled RNAidropout viability screens were performed using a 27,500 shRNA librarytargeting 5000 genes in PDX-T1, a TNBC PDX (FIG. 15 ). The screens wereperformed in two different formats, in vitro as organoid cultures and invivo as PDXs in nod scid gamma (NSG) mice (FIG. 1A). The abundance ofindividual shRNA in each experimental sample and the baseline referencesamples were determined by high throughput sequencing of the shRNAbarcodes. The goal was to identify genes whose knockdown by shRNAinhibited the growth of PDX tumor cells across different experimentalconditions. Consistent with screens in other tumors, the in vivo screenhad a more significant shRNA dropout rate (FIG. 5A) compared to the invitro screen (FIG. 5B). FIGS. 5A and 5B are graphs showing the shRNAcounts in the in vivo growth experimental sample (FIG. 5A) and in the invitro growth experimental sample (FIG. 5B) versus the shRNA counts inthe baseline sample. Control shRNA targeting luciferase (light graydots) and shRNA targeting CDK19 (dark gray dots) are highlighted. Allother shRNA are shown as black dots (each experiment performed once).The final candidate list was restricted to genes with the lowest 5% ofshRNA ratios in each screen that were targeted by more than two shRNAsand were also identified both in vitro and in vivo (FIG. 5C). Thisresulted in the identification of 46 candidate genes (FIG. 5D).

CDK19 was chosen because data from the Cancer Genome Atlas (TCGA) showedthat CDK19 copy number amplifications and mRNA upregulation were moreprevalent in TNBC patient samples (23%) compared to samples from otherbreast cancer subtypes (see, e.g., Cancer Genome Atlas Research, N. etal. The Cancer Genome Atlas Pan-Cancer analysis project. Nat Genet45:1113-1120, 2013; FIG. 6A). Additionally, high CDK19 expression hasbeen reported to correlate with poor relapse free survival in breastcancer patients (see, e.g., Broude et al., Current cancer drug targets15, 739-749, 2015 and Porter et al., Proc Natl Acad Sci U S A 109:13799-13804, 2012). CDK19 belongs to a subset of the CDK family that isreportedly more associated with regulation of RNA polymerase II (RNAPII)transcription than cell cycle progression. CDK19 and its paralog, CDK8,can both form the CDK module (CKM) by binding with three other proteins:MED12, MED13, and Cyclin C. The presence and nuclear localization ofCDK19 in our PDX cells were confirmed by immunofluorescence (FIG. 6B).In FIGS. 6A and 6B, the percentage shows the percentage of samples withCDK19 copy number amplifications or CDK19 mRNA upregulation intriple-negative, HER2 positive, estrogen receptor positive, and allbreast cancers. The fractions show the number of positive samples andtotal samples in each group. Data obtained from cBioPortal (see, e.g.,Gao et al., SciSignal 6, pl1, 2013).

4.3 Example 3 - Growth Inhibitory Effects of CDK19 Knockdown

To validate the growth inhibitory effect of CDK19 knockdown, threecommonly used TNBC cell lines: MDA-MB231, MDA-MB468, and HS578T wereused. Using two different shRNAs (shCDK19-1 (SEQ ID NO: 1) and shCDK19-2(SEQ ID NO: 2)) that independently target CDK19, the knockdown of CDK19(FIGS. 7A and 7B) was confirmed. For both FIGS. 7A and 7B, the relativeexpression of CDK19 in CDK19 knockdown cells is normalized to the meanexpression of CDK19 in cells transduced with control shRNA. Geneexpression in each condition is normalized to beta-actin as ahousekeeping gene (**P < 0.01; ****P < 0.0001, mean ± s.d., (FIGS. 7Aand 7B) n = 3 (FIG. 7C) n =2, experiments performed twice). Theknockdown of CDK19 also showed that it caused decreased proliferation inall three TNBC cell lines (FIGS. 1B-1D). FIGS. 1B-1D demonstrate thatCDK19 knockdown significantly decreased the viability of TNBC cells(viability of MDA-MB231 cells, ****P < 0.0001 (FIG. 1B), MDA-MB468cells, ***P < 0.001; ****P < 0.0001 (FIG. 1C), or HS578T cells, *P <0.05; ****P < 0.0001 (FIG. 1D) assessed 4 days after transduction withcontrol shRNA or CDK19 targeting shRNA (shCDK19-1, shCDK19-2)). Allvalues in FIGS. 1B-1D were normalized to control shRNA sample (mean ±s.d., n = 3, experiment performed twice, P values determined by unpairedt-test).

In the same TNBC PDX used in the initial dropout screen (PDX-T1), CDK19knockdown (FIG. 7C) also inhibited the formation of organoid colonies(FIG. 1E). In FIG. 1E, colonies were counted 2 weeks after transductionwith either control shRNA or CDK19 targeting shRNA (shCDK19-1,shCDK19-2), ***P < 0.001 (unpaired t-test) (mean ± s.d., n = 6,experiment performed twice). To determine the effects of CDK19 knockdownin non-transformed mammary cells, human mammary epithelial cells (HMEC)were infected with shRNA targeting CDK19. In HMECs, neither of the twoCDK19 knockdowns affected the viability of the cells (FIG. 1F). In FIG.1F, viability of HMEC cells was assessed 4 days after transduction withcontrol shRNA or CDK19 targeting shRNA (shCDK19-1, shCDK19-2). Allvalues are normalized to control shRNA sample, ns is P > 0.05 (mean ±s.d., n = 6, experiment performed twice, P values determined by unpairedt-test). Collectively, the studies show that in vitro, CDK19 knockdowninhibits the proliferation of multiple TNBC cell lines and the formationof PDX organoid colonies but does not adversely affect the growth ofnon-transformed mammary epithelial cells.

We extended our studies to more physiologically relevant in vivo systemsby knocking down CDK19 in three different TNBC PDXs grown in NSG mice.These PDXs: PDX-T1, PDX-T2, and PDX-T3 were derived from chemotherapynaive patients (FIG. 15 ). In these studies, all PDX tumor cells werefirst labeled with green fluorescent protein (GFP) and cellssubsequently infected with either CDK19 shRNA or control shRNA wereadditionally labeled with red fluorescent protein (RFP). Measuring thepercentage of GFP-labeled tumor cells that were also RFP positiveallowed us to determine the effect the shRNA had on the PDX tumor cells.With each of the two CDK19 shRNAs tested, CDK19 knockdown led to asignificant reduction in the percentage of RFP positive cells in tumorsfrom all three TNBC PDXs (FIGS. 1G-1I and FIG. 1M). Tumor growth wasmonitored and tumors were analyzed when they exceeded 17 mm. Thepercentage of RFP positive cells in PDX-T1, ***P < 0.001; ****P < 0.0001(FIG. 1G), PDX-T2, ****P < 0.0001 (FIG. 1H), PDX-T3, **P < 0.01 (FIG.1I), or PDX-T4, **P < 0.01 (FIG. 1J) were determined by flow cytometryand normalized to the mean RFP percentage of the control shRNA samplethat was set to 100%. Each data point represents one mouse. For FIGS. 1Hand 1H, mean ± s.d., n = 9, experiment performed three times. For FIGS.1I and 1J, mean ± s.d., n = 3, experiment performed once. For all, Pvalues determined by unpaired t-test).

FIG. 1M shows representative images of PDX-T1 tumors transduced withcontrol shRNA (top row), shCDK19-1 (middle row), or shCDK19-2 (bottomrow). Bright field images (left column) show gross tumor morphology,FITC images (middle column) identify tumor cells labeled with GFP andTexas-Red images (right column) identify shRNA-transduced cells labeledwith RFP.

These results confirmed that CDK19 is critical for tumor growth in vivo.CDK19 knockdown prevented transduced (RFP positive) TNBC cells frommetastasizing to the lungs in mice. Percentage of mice with RFP positivelung metastases from mice bearing PDX-T1 (FIG. 1K) or PDX-T4 (FIG. 1L)tumor xenografts are shown. Number of mice with RFP positive lungmetastases and total number of mice in each treatment group is shown asa fraction for each condition. PDX tumor cells were transduced witheither control shRNA or CDK19 targeting shRNA (shCDK19-1, shCDK19-2)(For FIG. 1K, n = 9, experiment performed three times; For FIG. 1I, n =3, experiment performed once). Furthermore, in PDX-T1, which normallymetastasizes to lung, CDK19 knockdown eliminated the detection of anylung metastases by those cells (FIG. 1K and FIG. 1N). In FIG. 1N, brightfield images (left column) show gross lung morphology, FITC images(middle column) identify metastatic tumor cells labeled with GFP, andTexas-Red images (right column) identify shRNA-transduced metastaticcells labeled with RFP. We also tested the effect of CDK19 knockdown onPDX-T4, an aggressive PDX obtained from the brain metastasis of apatient with a chemotherapy-resistant inflammatory breast cancer. Sinceinflammatory breast cancers are known to be aggressive, difficult totreat, and associated with extremely poor prognoses, it is notable thatCDK19 knockdown inhibited both the growth of the PDX (FIG. 1J) and thelung metastases in these mice (FIG. 1L and FIG. 7D). These data showthat in vivo, CDK19 knockdown not only affected primary tumor growth,but also inhibited tumor metastasis.

4.4 Example 4 - Identification of Tumor Initiating Cells (TICs) Withinthe TNBC PDXs

Given that CDK19 knockdown inhibited growth in two independent assayscommonly used to assess tumorigenicity (PDX growth in vivo and organoidcolony formation in vitro) and genes critical for tumor initiation arefrequently amplified or overexpressed in a subset of cancers, it ishypothesized that the tumor initiating cells (TICs) might be sensitiveto CDK19 inhibition. Thus, we sought to identify the TICs within theTNBC PDXs. Previously, EpCAM and CD49f were utilized to isolate cellsub-populations in normal breast tissue and in breast cancers. However,in many TNBC PDXs, EpCAM and CD49f often cannot clearly separate cellsinto distinct sub-populations (FIG. 2A, left). Thus, we utilized thebasal cell marker, CD10 with EpCAM to FACS-sort breast cancer PDXs. Wediscovered that CD10 and EpCAM can separate PDX cells into threedistinct sub-populations, EpCAM^(med/high)/CD10^(-/low),EPCAM^(low/med)/CD10^(low/+), and EpCAM⁻/CD10⁻ (FIG. 2A, right). In FIG.2A, the large inseparable cell population (left) seen using EpCAM andCD49f, becomes three distinct sub-populations using EpCAM and CD10(right): EpCAM^(med/high)/CD10^(-/low) (gate (1)),EPCAM^(low/med)/CD10^(low/+) (gate (2)) and EpCAM⁻/CD10⁻ (gate (3)). Theoverlap of these three sub-populations using EpCAM and CD49f is alsoshown (FIG. 8A).

To test the tumor initiating capacity of the three EpCAM/CD10 separatedsub-populations, we performed organoid colony formation assays in vitroand transplantation limiting dilution assays (LDA) in vivo. In organoidcolony forming assays, the EpCAM^(med/high)/CD10^(-/low) cells formedsignificantly more organoid colonies than theEpCAM^(low/med)CD10^(low/+) cells (FIG. 2B). In FIG. 2B, theEpCAM^(med/high)/CD10^(-/low) cells formed significantly more organoidcolonies than the EPCAM^(low/med)/CD10^(low/+) cells, *P < 0.05(unpaired t-test) (mean ± s.d., n = 3, experiment performed twice). Intransplantation assays performed in NSG mice, injection ofEpCAM^(med/high)/CD10^(-/low) cells from all six PDXs consistentlyformed tumors (FIG. 2C), sometimes with the transplant of as little as100 cells (PDX-T1 and PDX-T2). In contrast, transplant ofEPCAM^(low/med)/CD10^(low/+) cells only formed tumors in two PDXs(PDX-T1 and PDX-T2), and only when transplanting high cell numbers (i.e.2500 cells) (FIG. 2C). Furthermore, no tumors formed from the transplantof EpCAM⁻/CD10⁻ cells from any PDX. Hence, TIC’s are enriched in theEpCAM^(med/high)/CD10^(-/low) sub-population of all PDX breast tumors weexamined.

Having identified these distinct subpopulations, we next investigatedwhether CDK19 expression was enriched in the more tumorigenicEpCAM^(med/high)/CD10^(-/low) cells compared to the less tumorigenicEPCAM^(low/med)/CD10^(low/+) cells. In three of the four PDXs examined,CDK19 expression was higher in the more tumorigenicEpCAM^(med/high)/CD10^(-/low) cells compared to the less tumorigenicEPCAM^(low/med)/CD10^(low/+) cells (FIGS. 2D-2G). To generate the datain FIGS. 2D-2G, relative expression of CDK19 in theEPCAM^(low/med)/CD10^(low/+) and the EpCAM^(med/high)/CD10^(-/low) cellsas determined by RT-qPCR. Gene expression in each condition isnormalized to beta-actin as a housekeeping gene. Relative expression ofCDK19 is normalized to the mean expression of CDK19 in theEPCAM^(low/med)/CD10^(low/+) cells. *P < 0.05 (unpaired t-test) (PDX-T1:mean + s.d., n = 2; PDX-T2: mean + s.d., n = 6(EpCAM^(low/med)/CD10^(low/+)) and n = 3(EpCAM^(med/high)/CD10^(-/low)); PDX-T3: mean + s.d., n = 6(EpCAM^(low/med)/CD10^(low/+)) and n = 3(EpCAM^(med/high)/CD10^(-/low)); PDX-T8: mean + s.d., n = 3. Allexperiments performed at least twice). Thus, while CDK19 was expressedin all the PDX tumors we examined, it was expressed at higher levels inthe more tumorigenic EpCAM^(med/high)/CD10^(-/low) sub-population inthree of the four tumors that we investigated.

To determine tumor initiating frequencies in the setting of CDK19knockdown, we performed LDA using PDX-T1 cells transduced with adoxycycline-inducible CDK19 knockdown construct to produceinducCDK19KD-PDX-T1 cells where we can control CDK19 expression (FIG.8B). In FIG. 8B, the relative expression of CDK19 in doxycycline treatedinducCDK19KD-PDX-T1 cells is normalized to the mean expression of CDK19in control inducCDK19KD-PDX-T1 cells. Gene expression in each conditionis normalized to beta-actin as a housekeeping gene (*P < 0.05, mean ±s.d., n =2, experiments performed twice). By comparing the in vivotransplantation of inducCDK19KD-PDX-T1 cells in the presence ofdoxycycline (+Dox) with inducCDK19KD-PDX-T1 cells without doxycycline(No Dox), we find that CDK19 knockdown eliminates tumor formation in allthe cell transplantation conditions examined (FIG. 8C).inducCDK19KD-PDX-T1 cells were injected into the mammary fat pads of NSGmice at 50, 250 and 1250 cells. Mice in the doxycycline group were fed adoxycycline containing rodent feed to induce CDK19 shRNA, while mice inthe control group were fed a normal rodent diet. Tumors were detected bypalpation of tumors. The number of tumors that formed and the number ofinjections that were performed are indicated for each population.Populations and injections where tumors formed are bolded (n = 5 pergroup) in FIG. 8C. Using ELDA, we discovered that the tumor initiatingfrequencies significantly decreased from 1 in 342 cells (95%Cl: 1 in 828to 1 in 142) in the control (No Dox) group to 1 in ∞ cells (95%Cl: 1 in∞ to 1 in 2587) in the CDK19 knockdown (+Dox) group (FIG. 8D). Both thesignificant decrease in tumor initiating frequency caused by CDK19knockdown and CDK19′s higher expression in the TIC sub-populationsuggests that TIC inhibition is likely responsible for the impairedtumor growth observed with CDK19 knockdown.

4.5 Example 5 - Identification of Genes and Pathways Regulated by CDK19

There is an 84% amino acid sequence homology between CDK19 and its welldescribed paralog, CDK8 (FIG. 9 ). CDK8 has been shown to play a role ina variety of malignancies including colon cancer, acute myeloidleukemia, and melanoma. Higher expression of CDK8 has been associatedwith worse prognosis in colon cancer (Firestein et al., Nature455:547-551, 2008). CDK8 knockout in embryonic stem cells was shown toprevent embryonic development (Porter et al., Proc Natl Acad Sci USA,109:13799-13804, 2012) due to its essential role in the pluripotent stemcell phenotype. The known cancer-relevant activities of CDK8 may includepositive regulation of Wnt/β- catenin pathway, growth factor-inducedtranscription, and TGFP signaling. Depending on context, CDK8 has alsobeen shown to either negatively or positively regulate transcription.However, recent evidence has suggested that CDK19 may functiondifferently from CDK8. In vitro studies showed that CDK19 and CDK8participate mutually exclusively of each other in binding to other CKMcomponents, while gene knockdown studies in cell lines of cervicalcancer and colon cancer showed that CDK19 and CDK8 regulate differentgenes. Our goal was to investigate in TNBC whether CDK19 and CDK8 havedistinct biological functions by examining global gene expressionchanges resulting from targeted knockdown of CDK19 or CDK8.

To understand whether the molecular targets of CDK19 in TNBC are uniquefrom CDK8, we knocked down each gene in MDA-MB231 and examined therespective gene expression changes relative to control. Overall, CDK19knockdown affected 3909 genes and CDK8 knockdown affected 4233 genes(FIG. 3A). However, only 12% of upregulated and 5% of downregulatedgenes in the CDK19 knockdown experiment were also affected by CDK8knockdown. This suggested that CDK19 and CDK8 largely regulate distinctgenes (FIG. 3A).

Gene set enrichment analysis (GSEA) of the CDK19 and CDK8 knockdowngenes allowed us to identify enriched Hallmark gene sets amongst themost upregulated or downregulated genes (FIG. 3B and FIG. 10 ). In FIG.10 , the Hallmark gene sets uniquely enriched in the knockdown of CDK19or CDK8 are shown in black, enriched in both the knockdown of CDK19 andCDK8 are marked by “*” and enriched by genes expressed in oppositedirections between the knockdown of CDK19 and CDK8 are marked by “**”.Normalized enrichment scores and FDR q-value are determined by the GSEAsoftware. An FDR cutoff of < 0.25 was used to select significantHallmarks. These Hallmark gene sets consist of genes that arespecifically involved in certain biological states or pathways. Genesassociated with known breast cancer-related Hallmarks such as mitosis(E2F targets, G2M Checkpoint, Mitotic Spindle), PI3K-AKT-MTOR signaling,MYC pathways (Myc Targets v1), glycolysis, apoptosis, and oxidativephosphorylation were changed in the same direction by CDK19 and CDK8knockdowns (FIG. 3B, middle overlap region), demonstrating aco-regulatory relationship between CDK19 and CDK8. Further, genesassociated with early estrogen response, epithelial to mesenchymaltransition (EMT), cholesterol homeostasis, MYC pathways (Myc Targetsv2), interferon alpha response, and fatty acid metabolism changed in theopposite direction in response to knockdown by CDK19 compared to CDK8(FIG. 3B, boxes), which suggests a counter-regulatory relationshipexists between CDK19 and CDK8. Hallmark gene sets enriched by theexpression of genes in opposite directions by CDK19 knockdown comparedto CDK8 knockdown are boxed. A number of the Hallmark gene sets wereonly enriched in the genes that uniquely changed due to CDK19 knockdown(FIG. 3B, left region). Hallmarks reflected by these gene sets includedP53 signaling, KRAS signaling, androgen response, NOTCH signaling, TGFBETA signaling, and IL6-JAK-STAT3 signaling, which may be potentialbiological pathways for targeted therapies for TNBC. All of thesebiological pathways represent active areas of clinical investigation inthe evaluation of targeted therapies for TNBC. Consistent with ourfindings, a number of the pathways found enriched in our CDK19 knockdownexperiments, such as cholesterol homeostasis, P53 signaling, mitosis,and NF_(K)B pathways have been shown previously in other cell types toalso be regulated by CDK19.

In summary, these analyses showed that CDK19 and CDK8 have the potentialto co-regulate certain pathways, while counter-regulating others.Furthermore, CDK19, like CDK8, is capable of positively or negativelyregulating biological pathways. The multitude of clinically relevantTNBC pathways regulated by CDK19 suggests that targeting CDK19 canprovide the opportunity to modulate multiple pathways simultaneously andat the same time, avoid potential toxicity because of the advantageouslimited tissue distribution of CDK19. This approach could overcome theresistance to single agent therapy commonly seen in TNBC and alsopotentially enable the targeting of ‘undruggable’ processes such asthose involving P53 or MYC.

4.6 Example 6 - Effects of CDK19 and CDK8 on Epigenetic Modifications

Recent studies have highlighted the role of CDK19 and CDK8, as well asother transcriptional CDKs (CDK7, CDK12/CDK13), in regulating thetranscription of critical oncogenic genes by acting at large clusters ofenhancers (also called ‘super-enhancers’) that are marked by histone 3lysine 27 acetylation (H3K27Ac). The exact mechanism for this generegulation is unclear, but is believed to occur in part throughinteractions of the CKM with Mediator to regulate RNAPII-Mediatorinteractions and in part by phosphorylating serine residues in theC-terminal domain of RNAPII. Given the propensity of transcriptionalCDKs to function at enhancers, we wanted to investigate whether CDK19and CDK8 can also regulate the epigenetic modifications at enhancersites as a mechanism to control gene expression. While enhancermodification through other signaling pathways have been identified, thismechanism of gene control has not yet been reported for the CDKs.

To explore the role of CDK19 in epigenetic regulation, chromatinimmunoprecipitation and sequencing (CHIP-Seq) for the H3K27Acmodification was performed on MDA-MB231 cells under three differentconditions: Control (empty vector transduction), CDK19 knockdown, andCDK8 knockdown. Genome-wide analysis of all H3K27Ac modified regionsshowed that both CDK19 knockdown and CDK8 knockdown had similar globalH3K27Ac levels compared to control (FIG. 11 ). In FIG. 11 , H3K27AcCHIP-Seq signals across all identified H3K27Ac peak regions arenormalized to 1-Kb and centered on the middle of those regions. Signalsof the flanking 2-Kb regions are also shown. To compare relative signalchanges, the total signal of each biological replicate was determined bysumming the signals of each 50-base window 1-Kb around the center ofeach region. P-values between total CHIP-Seq signals of each sample weredetermined by unpaired t-test. Through comparative analysis of H3K27Aclevels in the CDK19 knockdown compared to the control, we identified3034 peak regions with increased H3K27Ac signal (All-H3K27UP) and 502peak regions with decreased H3K27Ac signal (All-H3K27DOWN). By excludingpeak regions that were also different in CDK8 knockdown compared tocontrol, we identified 2309 peak regions with increased H3K27Ac signal(CDK19KD-H3K27UP) and 432 regions with decreased H3K27Ac signal(CDK19KD-H3K27DOWN) that were unique to CDK19 knockdown. The specificityof these regions for CDK19 was investigated by comparing the H3K27Aclevels at these regions in CDK19 knockdown, CDK8 knockdown, and control.Compared to control, enrichment of H3K27Ac levels across theCDK19KD-H3K27UP regions (FIG. 3C) and depletion of H3K27Ac levels acrossthe CDK19KD-H3K27DOWN regions (FIG. 3D) were significant only for CDK19knockdown and not for CDK8 knockdown. In FIGS. 3C and 3D, ***P < 0.001;ns is P > 0.05 (all samples n = 3, experiments performed three times).H3K27Ac CHIP-Seq signals of the CDK19KD-H3K27AcUP or CDK19KD-H3K27AcDOWNregions are normalized to 1-Kb and centered on the middle of thoseregions. Signals of the flanking 2-Kb regions are also shown. To comparerelative signal changes, the total signal of each biological replicatewas determined by summing the signals of each 50-base window 1-Kb aroundthe center of each region. P-values between total CHIP-Seq signals ofeach sample were determined by unpaired t-test. Thus, CDK19KD-H3K27UPand CDK19KD-H3K27DOWN define peak regions where the H3K27Ac signal ismore specific for, and most sensitive to, knockdown of CDK19 compared toknockdown of CDK8.

We next assessed whether increases or decreases in H3K27Ac levels as aresult of CDK19 knockdown corresponded to changes in gene output. Forthis, the previously defined All-H3K27UP and All-H3K27DOWN peak regionswere annotated by proximity to the nearest gene to establish two genesets: CDK19KD-EnhancerUP (1593 genes) and CDK19KD-EnhancerDOWN (341genes) for further analysis (Table 1 and Table 2). GSEA of these genesets with our CDK19 knockdown gene expression data indicated that genesmost upregulated by CDK19 knockdown were enriched for theCDK19KD-EnhancerUP genes (NES 1.68, FDR q-value = 0.000) (FIG. 3E),while genes most downregulated by CDK19 knockdown were enriched for theCDK19KD-EnhancerDOWN genes (NES -1.84, FDR q-value = 0.000) (FIG. 3F).Thus, as a result of CDK19 knockdown, perturbations to the H3K27Acsignal at the putative enhancer elements of genes correlated well and inthe expected direction with changes in gene expression.

TABLE 1 CHIPSEQ_CDK19-KD ENHANCERDOWN NDRG3 TTLL11 CYB561 KAZN PPM1ASLC25A32 GRAMD4 S100Z SNRK YWHAZ FAM168A KIAA1524 CDH4 PAQR5 KCNK12NSMAF RNF169 SLC35F3 HDAC8 KCNAB1 CDKAL1 ZFYVE9 AK7 DDX31 WDHD1 RNF144BDGKB FKTN C6orf203 EPB41L2 RUNX2 CXCL8 PLXNA4 TOX2 XPO6 PGM2 TRIM60 PKP2TWSG1 RGCC AZU1 NORAD ARFIP1 SSH2 ALKBH8 TMBIM4 IPO5 TTC39C KITLGC11orf87 SCN5A ZCCHC24 FBXO11 RAI14 ABCA8 PRNP OC90 STX8 LOC341056 MAGT1FOS LPA OPHN1 FGF9 MPP4 IQCJ RPL7L1 ZFAT ABCA13 CSGALNACT2 KIAA0586RNF114 TOX G6PC2 BACH2 RGMB C1QTNF3 MOK MED27 WWC1 SPRED1 C11orf63C12orf75 HRH1 NTNG1 GGCX ADCK2 PDE7B UBASH3B ZNF281 LOC100506797SLCO4A1-AS1 WDR27 RBM5 AKR1B15 ENKUR CACNA1A WDR89 SLIT2 SHTN1 ALK TLE1FAM107B ELOVL5 FZD8 CSTF2 XRRA1 ARSF STX18 KIF3C SLC25A12 HIVEP1SATB2-AS1 SNX14 IDNK OXCT1 ZNF133 TAPT1 STK38 STRA8 TMEM18 UTP18 VAPACCR1 SPPL3 MBP ASAP3 SEMA4D TBL1X SMYD3 ITGB1BP1 CRTAM MDM1 TRHR FAF1STK4 SMIM19 DNAJA3 PDE8B TSNARE1 KCNV1 AVEN FAM20B CDH13 KIAA1109 KHDC1DAP HIPK3 OR10V1 VTI1A FIP1L1 AKAP1 C20orf85 PPP4R1L IL10 PIK3CB ALG10BATAD1 ZBTB10 TNRC6A COMMD2 MLEC NCK2 FAM171A1 SGPL1 NFATC1 GRB10 NECAB1AMOTL1 RHOH HDAC9 PDE4B RFX8 NR2F1-AS1 RNF34 TMSB10 KYNU TMEM235 SLC26A8SIK3 CHI3L2 PPP3CA HESX1 CORIN ARHGAP18 SYAP1 OLIG2 THG1L MAST2 PPA2BTBD9 GPR68 EPB41L1 OLFML2A CFAP36 KLHL5 PRDMS COMMD7 CEP112 SVILC1orf21 PUM2 ST3GAL6 MTCL1 RPAP2 ATG5 PLEKHM3 EDEM3 SAP18 PANX1 MAB21L2PTPN20 DSCR9 SIPA1L1 SUMF1 CDK5RAP2 UBR5 GBF1 UBE3A INHBC EPS15L1 CD226TCF7 TGFBR2 HTR7 BCAP29 PRLR USP43 ATP6AP1L RPS6KA5 EXOSC7 RAB10 KCNG1CPD KIAA1147 RPS3A CCDC152 ATF7IP CCDC88A CASS4 ADM2 GTF2H5 FER1L6 DDR2PARD3 PREP RPL5 C1GALT1C1 GJD4 WWP2 SVIP FZD4 BPGM ARMC9 ERICH6B MAP1BTCP11 PLS3 NT5DC3 CBLN1 C5orf42 LIN7A FIBP TSEN2 CSNK2A1 UBE2V2 CMTM8ARHGAP25 KAT7 BLCAP IFI44 TMEM38B EDNRA LOC285696 GOLIM4 NEK1 C3orf67PRDM8 TBXAS1 SND1 ANAPC10 TSPAN9 ARC ETV1 CTDSPL NDRG1 WWTR1 WASF2 ADH7NNT SLC46A3 CTNND2 MBD2 HYPM RNF217 CHST11 CLDN2 STAG2 INTS6 ZMIZ2 CHSY3MRPS28 CBFA2T2 BTD CEP290 RIN2 COX7A2L TMEM30B WASF3 APCDD1L PARP12FAM46C TCF12 FKBP1A ARFGAP2 PUDP LDHD ADGRL3 TMEM50A TRDMT1 TSEN15 BAZ2ATANC1 NANS TAOK1 MAPK8 PPP4R3B FAM196A OAT AGA DNAH6 ARHGEF4 PSMC4ANTXR2 BASP1 TPTE2P1 OR2AT4 MMAB DENND2D C7orf73 ST18

TABLE 2 CHIPSEQ_CDK19-KD ENHANCERUP HLCS EFCAB13 FBXL20 AGR2 ABCC11MFSD7 RIC8B KCNT2 IGF1 SLC12A8 AZIN1 LYSMD4 AVIL ATP2B4 ASS1 MARCKSL1CDYL CRABP2 ERCC8 OSR2 CASQ2 ACTL7B TNFRSF11A NAV2 LHFPL2 TEX35 SLC22A16LUM PRKCZ RDH16 ERICH2 STPG2 HGC6.3 PTPRE GPCPD1 BEGAIN BEST3 ABCG1ZFPM2 SOWAHC MYL4 TCF7L2 HAS2 IGSF22 BDKRB1 MYL12A DNAJB11 LOC10050679 7NNAT SCAF8 LOC10026816 8 PPP1R36 CDC42EP5 EDN1 SP4 SOWAHB NEURL1 TSC1MIS18A RALGPS1 SH3BP4 C15orf53 GJA4 FOPNL RPIA STOM VEGFA AHDC1 DBX1PHACTR1 ALDH1A3 DACT1 SLC1A2 SRPX2 PLXNA2 TBC1D14 RAD23B MAP1A ECHDC3GLI2 IQSEC1 ANKRD16 CHAT MAGEF1 NOL6 SUB1 RFK CHRNE DENND3 NEK6 S1PR1C12orf76 DIEXF DHRS9 ERICH5 SCCPDH TAF1B XPR1 RYBP ANP32C MCHR1 DLX4OSBPL11 ARHGAP12 FGD2 SNTG1 PTGER4 AGMO PTRHD1 FANCA AES KRBA2 ZC3HC1TRIM24 HMHB1 IRF2BP2 INPP5F CACNG2 HHLA3 CFI TTLL5 ACBD3 PLB1 EDIL3IGFN1 TROAP HAUS8 NOV HPSE2 YARS PROC LEPROTL1 EFHD1 GALNT12 KANK4 JAK3TMEM170 B DCLK1 PTPRN SPATA16 CCDC97 ZNF787 TPRG1 DAPK3 KIF25 LMCD1AADACL4 RFXAP ALOX5AP BIRC7 GBA3 C1R TMPRSS5 TMEM100 OR1M1 ENO2 PTPN3FAM196B CLEC14A TSPAN 1 NPC1L1 TBL1X PTPRR LOC10013087 2 FAM136A HSPH1STK17B GSTA3 ACKR3 OPTC CREB5 PHTF2 SMIM20 SPRED2 SHE AGAP1 CTAGE1KIF16B TRAF4 FAM57A KIAA1211L CORO6 SPNS2 MAOB SOAT1 TRIB1 KCTD4 CELF2TWIST2 C19orf38 TMEM40 THEM4 GSX2 ADAT2 USF2 NRP2 NSUN7 SEMA3E ZNF462SUGCT BCAT1 CSNK1A1 RAD51AP2 FFAR4 NINJ1 SHH SPIB PSAT1 CLDN1 ERGIC1SLC15A1 KISS1 C11orf49 NAT2 HECW1 EXOC6B KLHL31 YAE1D1 PIM3 DGKZ MEF2AUSB1 CAB39L PCSK1N MAST2 STON2 HIP1R ELF3 C4orf26 ZNF429 DISC2 CENPBMTCH1 PALLD GLRA3 ZSWIM3 NMBR C14orf37 GPR108 GSTP1 ATP1A2 RBM47 SORBS3RAB14 RPS29 ACVR2A C11orf94 DAW1 THADA CKAP4 SF3B5 ANO6 BTBD16 XRCC2OTOS EMX1 EPS8L3 PTK2 ZNF318 RTN2 CAMK2D MRPL4 VGLL3 LMNTD2 CAB39 PAPSS2TRIML1 ZSCAN18 HCAR1 RPS3A FAM81A FIZ1 NEK2 EHF NEDD4L SYT2 LEPROTMAP7D3 PRTFDC1 SEC14L5 HYI SLC44A1 BAG1 GFI1 MFSD4B GCG PPEF1 LAM B4NANOS1 YWHAEP7 ATG9B GCNT3 ATXN1 LIMD1 P2RY1 TMEM120B SLC37A1 GRHL3OTUD3 VWA2 IGFL1 P2RX7 TLR10 KIAA1324 MAPK8IP1 SLC2A8 RHOB CAMSAP2TMEM95 FAT1 TFAM APIP PPM1L ETS2 KPNA7 HRK ACOT11 RGS7 TMEM106B CERS4NXPH2 SLC30A6 RREB1 EML5 WFIKKN2 PAK1 FJX1 HMGCR RCAN1 GUCY1A2 LAMC3RBFOX2 BMP6 DSG3 PITPNM3 ISL1 PACSIN2 TSN BCOR HES1 NIPBL STAT4 CDH3PSG2 SLC39A10 XIRP1 NAB1 DYNC2H1 TMEM51-AS1 ARRB2 CCL20 MINK1 MRPL15LY86 PLEKHA1 M ETTL6 LRRC8D SPR SCRT2 RALA MAPK1IP1 L EGLN3 CRISPLD2PAPLN MOAP1 COL24A1 MYO5C SLC28A3 MAP3K7CL RB1CC1 SERPINB10 TPD52L1PPARA MZT1 ATP8B2 RASSF6 PIGU ADTRP CYP1B1 LRRFIP2 NLN ZC3HAV1L NECTIN1CELA2A SYT14 CDCA4 FBXO3 ASCC3 SH2B2 C3orf58 ENOX2 PLEKHG4 DAAM1 TINAGL1YIPF6 GPR135 ZNF160 ANXA1 ERCC3 SLC39A11 CDKN3 CBX4 RALGDS TUBA1A PMAIP1MN1 ADAMTS10 FGFR3 EPAS1 ZCCHC10 LRRC4C DUSP18 CXCR5 CRABP1 MAST3 ABLIM2INO80C TLR2 AKAP10 RASAL2 NR4A1 PNOC SCN3A NOCT DDC TACC2 IFNLR1 COL4A5FOXQ1 DSG2 PPFIBP2 MAD2L1 FILIP1L ASH2L TJP3 NID2 DAOA-AS1 CAPZA2 RMND5ASLC8A2 STC1 DDX47 RXFP3 COL6A3 PDE8A RGS1 TMEM119 MXRA5 KCTD16 WDFY3EMILIN2 PSAP SETBP1 GPRC5C MAST4 DNAH1 RPUSD4 KCNJ15 CCDC9 COX6B2 MEDAGIL6R NUAK1 ZP4 CD276 EVA1C DPEP2 ABHD5 MRPS22 GLDN RPH3AL AQP7 LRRFIP1GHSR NME9 SALL4 F5 MCOLN3 GPATCH1 VSTM2L PDLIM1 KIAA0753 STK39 TNFRSF11BHSPBAP1 SLC9B2 PEX26 CNGB1 CDKAL1 SLC34A3 KERA UBAP1 JADE1 IQCA1 FKBP6SARM1 DLX1 P3H2 ITPR2 BTBD10 FBLN2 HES2 C1orf100 KRT10 NEDD9 GATA6 PLAC8FAM198B FBP2 BSN SPINK2 PFKP C11orf88 SIN3B ORMDL3 TBX21 GPR173 KAZNKIAA0040 HDAC11 FAM96A DCHS2 UPF2 KCNMA1 PLA2G4E ARL4C HCN3 COL14A1BEST1 ACSBG2 NPFFR1 TMEM178A CDC123 NDUFA12 CDNF RBM45 CBLB PIM1 CTSODUSP6 LHFPL5 BCL2L10 DIXDC1 TCF12 TNFAIP8 PPM1H SMARCD3 RAD54B C4orf45CREB3L2 NPVF OR6B1 HMGCS1 SSR3 CXCL13 TTC8 MAPRE3 SLC2A6 SERPINB7 HTR3AUSP36 VIT C17orf99 CYP27C1 NFKBIZ AHCYL2 DHRS7C KRT32 COL19A1 NOL10 MUC1SYT17 GRHL1 DENND2C CLCA1 WNT11 INTS10 CRELD2 LGR6 FHL1 ARRDC5 PARVB CRKNECAP2 KRTAP4-5 CLMP NCF2 GGT7 INSR PIK3C2B C11orf65 CLUAP1 MBL2 ASPSCR1YBX3 SLC35F2 NEMP2 CLDN22 DAB2IP MAMDC2 IL37 CDCA7 GAREM1 DCN ATP12AREPS2 FAM216B CTTN KLHDC9 CDKL2 AGRN ATP9A OXT OLIG2 TSEN54 KIRREL2HECTD1 ME3 INTU PGRMC2 RFX2 DSPP LSM8 TEX9 MYEOV POLR1A PKIG NEBL SOX4HFM1 OSBPL5 TANK CALHM3 TLE1 TRIM66 SACS SLC10A7 MTM1 KLHL38 SHCBP1LSRP19 TMC1 TOR2A FNDC3B XIAP JARID2 ALG1L NYX BMP10 TRAF3IP2 WISP2 SCN1BC15orf56 ARHGEF3 EPB41L4A AFG3L2 MON1A PSMA6 TRIP13 ACTR10 GJD3 EFCAB11IRF4 SLC22A23 INPP4B HNF1B KIAA1522 ALPP KRT37 MMP24 CMTR2 ARHGAP29SSUH2 NUBPL RRAD CDH2 CREB1 ZNF621 APOBEC1 HIPK3 METAP1D SPOCK2 PTPN1INHBE ACHE UGP2 PITX2 RPS5 PTAFR P2RX4 GJB4 PRKCSH C12orf71 ZNF292 PKP1MAOA YPEL5 NKAIN1 KCNG1 EXD1 KRT39 STAU2 AFAP1L2 MIER3 ATG14 HRH1 CYB5BQPCT TRAK2 IL12B AP4S1 ACSL1 LAMA3 GJA1 OR51B6 FOXS1 RPTOR VAPA ASIPSPIN1 ZNF542P THRB COX11 RPS23 PIGC CREBL2 PIN1 UNC13A GPBP1 ATOH8PPFIA2 DMKN ZBTB43 INPP5K STRA6 ABR DEF6 DACH1 LILRB3 POLE4 CAPZA3 SNX13MMP16 MOGAT2 NRCAM NRARP GATA2 TMEM65 FBN2 INTS7 INPP4A TMEM38B C10orf67ATXN7L1 GPM6A GSTZ1 GARNL3 USP38 AKTIP NR2F1-AS1 KMT5B LGALS9 ZFP36L2CD200R1L GPATCH2 DLL1 CNGA2 GAS7 RIPPLY3 FAM161A C10orf113 TRPC4 RAB27BCD109 TNS3 CDHR2 TNFRSF21 FAM50B CAGE1 RNF220 ARFGEF3 RALB INTS1 VWA3BSLC30A1 ITCH UPP2 LZTS3 YTHDF1 AKR1D1 TAS2R16 DPF3 IL15 LGMN ST8SIA4PROSER2 SHC3 PLEKHH1 GRIN2D CCDC184 PLEKHG6 METTL25 CYP26B1 SHC4 GSG1LBMPER C3orf38 STEAP4 FA2H TMEM88B PPARGC1A IGFBP3 HSD17B14 RNF112 CXCR4TESC AHR CDK14 CD36 CLTC TFCP2 PRRX2 FOXD2 ATP6V1H GALNT15 FSCB YTHDC2C10orf35 ZNF92 COMMD1 0 RPTN RGS11 C9orf135 IFT81 TTI1 AMTN LPIN1 IRS2SLTM MYO1G FGFBP1 LRRC25 RPS6KA3 BCR EEPD1 RSPH1 LAMC1 KLF5 HRASLS2FOXN3 ANO10 GTF2E2 TRIM9 PAPPA RABEP2 PCLO ATF3 PAFAH1B2 PRSS57 FAM3BCYP24A1 CARTPT NPEPPS NTF3 TLR3 CABLES1 SLN ANGPT1 TUFT1 CLCNKA ANXA4NCALD LTBP1 CPPED1 DYNLT1 MREG C4orf19 AIG1 THNSL2 RBM3 SMG6 OR2S2 AMER3NAV3 RNF13 PPP1R2 SLC43A3 WIPI2 PANK1 TAMM41 ST6GALNAC4 TRIM54 NAT1PTPRC COX20 CCDC65 NRDE2 SPAG9 DCTN6 GABBR2 UPP1 ARHGEF18 C1orf226TTC39C TGFB3 EIF4A2 UNC5A TPK1 LBH PRKAR1A SERBP1 TOPBP1 LARP4B SERPINB1C12orf74 GOSR2 OSCP1 PSTK KNOP1 C1orf228 CD9 ACSL5 CT62 DENND5A BAIAP3SLIT2 NFATC1 DNAJC6 ADAMTS6 ADAM29 FRMD3 RAB8B OR10H1 RAB11FIP 4 TMEM45BFABP3 NNMT WDFY1 CEP152 ARHGAP42 HIC1 SHQ1 TARBP2 SNX7 C10orf90 RAD51BLRRC20 GNLY TIMM22 SMARCA2 DUSP8 SOX8 NCOA6 CCNY COX7A2 TRAF3IP1 SLC6A3KBTBD12 ARHGAP39 MDFIC PPM1B SEMA3A PLA2G2E DNAH11 EGR4 DUSP27 TMPRSS7NFE2 CACUL1 TMEM86B TRABD2A REEP3 NDUFB11 CCDC186 C7orf57 CC2D2A LEKR1ATP6V1G1 MPZL2 SLC4A1 MGAT4C TMPRSS9 COL21A1 LBP TMEM247 CCDC34 IGF2RCLDN4 WNT7A APBB1IP CPA4 DTWD1 NSMCE4A GDPD5 ANKRD33 PLEKHG3 CYP11A1CDCA7L NID1 MAF NUP155 CAMK2B ZEB2 ARID5A FRMD4B ATXN7 LSM3 FGGY ABI3BPPNPLA5 ATXN3L ZNF396 SOCS2 M BTPS2 HS1BP3 C9orf3 MUC20 IL7R FIGN PPP2R2CUSP2 ENKUR RALBP1 MRPS18A CNIH3 ULK1 ADGRF1 FLJ23867 CRIP2 PTHLH FAM187BSH2D3C SH3GL3 ODC1 LGALSL PRRC1 GC NEK10 MAMLD1 C4o rf3 2 SH3TC1 LGI1SLC6A20 CD180 PLCE1 THY1 CTSH SLCO4A1 SLC26A9 MPL AACSP1 COL26A1 SSR2CMAHP ID4 ALCAM TAGLN2 COBL SCNN1G TRAF7 MYOZ1 AKR1C3 CER1 AREG ABCG2DCK CCDC174 PRKCE CBLC SYNM BCAS2 BDKRB2 NABP1 TBC1D1 DHRS3 TES USHBP1UBQLN4 ETV7 CCR8 AGPAT2 MLXIPL SLC13A1 ADAD1 NOSTRIN QRFPR RHOBTB1 SCFD2PPP4R3A E2F6 CDK4 PABPC1P2 COL5A3 RAB31 PPP1R14D CASK ADAMTS15 CRTAC1HRC KCNQ4 UBE3D MIEN1 KIF18A BPI KIAA0895 SIM2 LITAF RNF165 CCDC77 DIO2ABCA6 ZNF473 FHAD1 BRINP1 GRAMD1C TAF1L EMC7 TRIM29 AGXT2 CD300LFPPP1R12B GPR37L1 WAPL AQP3 LZTFL1 YIPF5 ENOX1 ZC2HC1A GIN1 FHDC1 PBOV1DERA FGD4 TYK2 ACP6 NLGN1 ULK4 BANK1 PER1 ITGA2 LLGL2 ALDH8A1 FBRSL1TPPP3 TNNI2 TMEM167A RGS4 PDGFB ZDHHC17 APOBEC2 THBD HGF BTN3A1EXOC3-AS1 NAA20 VAV1 ZNF664 TRMO TMEM139 PRR15 PHLPP1 GINS2 GMDS PCDH1PARD3B MYH13 C1orf43 ARSB TMEM217 SLC22A2 IL1RN FMN1 KCNJ12 RASAL3 HTR1BPCDH8 BRDT NEK7 MCM10 NPSR1-AS1 ARID5B SEMA3C UBB TACR2 VSX1 LOC100132215 MMD2 MEF2C SPON1 FLVCR2 SNX25 GLOD5 STK38L ZNF555 YKT6 NR5A1 DNAJC10SYNPO2L APEH ALDH3A1 DPYSL2 ETFB GCM2 FGF19 GRN GNAS FCHO1 DBN1 TCEANCSOCS6 CEP128 RBM24 HEATR5A ASAH1 CHMP6 RPS26 MRVI1 PLA2R1 CDC14B SCARB1SLC7A10 SLC13A2 WDR89 VPS45 INSIG2 GJA3 MCM5 TRPS1 CHMP4B ZNF366 SRMSCNTN6 MYO5B AGTPBP1 TMC8 FAM173A PITX3 TRAFD1 PNPLA8 CD28 YWHAQ C9orf116SLC16A3 VPS37D ASB5 JSRP1 UGT8 WBP2NL TSPAN2 EGFR SRD5A3 CDC16 NDUFA10SPOPL NR5A2 ZC3H4 KLF4 C9orf153 GADD45A C18orf12 EMX2 BMF PPP2R5A MKL2TIMM17A CMIP METTL4 FEM1C ST6GALNAC 5 PIWIL3 SRL CCBE1 CIT ASB7 C15orf54TMEM71 TGIF1 ARVCF MEGF6 TPPP TNFRSF19 RAB11FIP1 MRPS36 FTH1 ETS1 MAN1A1PELO OXER1 DYM SLC23A3 MMP20 KCMF1 TRY2P RPS6KA5 NPAS2 SLC25A19 CCDC112SOX9 RGS9 NUTF2 OXSR1 MAGEB2 AVP TMEM59 C9orf50 ABHD11-AS1 GPR132 PLCD1NATD1 OTUD1 PLA2G4D BHLHE41 AAED1 TMIE NDUFB6 SPCS3 PRRG4 GCLC CEACAM22PLIN28A KIF5C PHLDB1 E2F8 EPHA5 CITED2 SLC5A1 TBC1D23 PLEKHG4B BANF2GLP2R HSD17B2 PTPRK SLC7A7 SLC9A7 SNX9 SND1-IT1 OLA1 PEBP1 TAPT1LOC401052 CLIC5 CPEB4 KDM4C SLC20A1 RAPGEF2 SGK1 TANGO6 SNCB SEMA3DFLRT2 NTRK2 LEPR C9orf131 IFI6 LVRN ZNF214 C14orf2 SSFA2 PABPC4L TMEM244C1QTNF1 TMC5 WDR18 BRMS1L CTNNB1 PDE1A SH3PXD2B NTN4 LIMCH1 PSD3SLC38A11 HTRA1 DIRAS1 EPHB6 HTRA3 PTGIR YY1AP1 TFAP2A GTPBP4 ARFGAP3LDHAL6A ZNF331 EPC1 SNRPC CREG2 ZBTB7C CDK20 KIAA0825 RXFP2 GPR182 CASZ1ZBED2 ASAP2 INPP5A UBE2O WNT7B TNFRSF8 RANBP3L SORBS1 GUSB CFAP126 SNHG7COL18A1 CACNA1A FKBP8 TEKT3 RPEL1 GNAT2 FAM107B LCA5 MAP1S RHOD ADSSL1SLC8A1 PKP2 ABHD15 FAM86B3P SNRNP35 SLC1A4 CLDN23 INHBB FAM110B TMEM207HMCN1 ADAM12 PRF1 CD38 METRNL OPCML RAP1GAP2 IQUB TP63 RECQL5 PIK3R1KRT20 CYP1A1 DUSP14 FTHL17 EPYC CCDC134 B4GALNT2 FOXE1 ADAMTSL1 SCINPOPDC2 NXNL1 RFX7 VTCN1 CPA2 IL21 PPP2R2A RAPGEF4 ARNT2 GSN SIGLEC8LRRC29 ZNF385B NLRC5 FUZ CCR3 VLDLR MELTF BDNF ACSL3 ZNF488 FRAT2 BATF3C11orf96 SULT4A1 ITGAV ADGRL3 SKIL SIRT4 MORN3 RIPK2 KLLN MYO6 MTCL1UBA7 JPH2 DYSF TYROBP CCDC83 RHOU NFIL3 FKBP11 LRPAP1 CLDN10 ERP44 IPMKLTBP4 BBS10 RNLS SPAG17 YOD1 BPTF FERMT2 SYT12 CCDC150 S1PR2 PRSS41FAM120B TPH2 CDKL3 SFXN4 NDRG4 FAM171A1 ANKRD10 SLC29A3 IRAK3 KCNA10ZBTB16 MICAL3 C5orf51 NAA16 EDNRA PRMT9 DCST1 PDC VCL RAD51C OSER1 SFRP2VSTM5 BCLAF1 CXCL16 BFSP1 SHISA2 LGALS3BP SCG2 TYMP NENF TEX36 C17orf107ST6GALNAC 1 C5orf30 KCNJ6 AGTR2 SHANK2 GPR156 MICALL1 ZNF608 CCDC63 AQP9MSX2 GPC1 GFPT1 GPRC5B LACC1 NPFFR2 FBXO7 PARP11 TIGD2 ANKRD9 LRRN3UBASH3A CCDC68 TDRD7 ARHGAP24 SH3BGRL2 PNMA2 SLC1A3 ABCA13 CIPC SPIRE2H3F3C EFHC2 VILL CACNA1H KCTD12 UBE4B NYAP2 DUSP23 CCDC124 RHOBTB2 ERBB4RAB35 ITPK1 PIK3R3 SPTSSA MMP27 UBASH3B PYM1 SPAG16 TOMM5 TLE6 MRPL21JPH1 PKD1L2 TMEM94 LANCL3 IL2RG FUNDC1

The aforementioned GSEA also enabled us to identify the leading edge‘core’ genes that contribute the most to each enrichment (FIGS. 12A and12B). At these ‘core’ genes, differences in H3K27Ac enhancer signals dueto CDK19 knockdown (FIGS. 13A-13D) result in large corresponding changesin gene expression (FIG. 13E). The gene tracks at the ELF3 (FIG. 13A)and ETV7 (FIG. 13B) loci show enrichment of H3K27Ac signals in the CDK19knockdown samples, whereas the gene tracks at the CHI3L2 (FIG. 13C) andCRTAM (FIG. 13D) loci show enrichment of H3K27Ac signals in the Controlsamples. Upper tracks denote Control samples, while lower tracks denoteCDK19 knockdown samples. Gray bars denote regions identified by DiffBindto be different between control and CDK19 knockdown samples (FDR <0.05). Metascape analysis was then used to evaluate Hallmark gene setsenriched within the CDK19KD-EnhancerUP ‘core’ and theCDK19KD-EnhancerDOWN ‘core’ genes. Within the CDK19KD-EnhancerUP ‘core’genes, early Estrogen Response (p-value = 8.72e-5) and EpithelialMesenchymal Transition (p-value = 1.08e-3) were Hallmarks identified asenriched (FIG. 3G, dark gray bars). Similarly, within theCDK19KD-EnhancerDOWN ‘core’ genes Androgen Response (p-value = 1.89e-3)was the Hallmark found to be enriched (FIG. 3G, light gray bar). Thus, asubset of genes (FIG. 3G) within the early Estrogen Response, Epithelialto Mesenchymal Transition, and Androgen Response gene sets have changesin H3K27Ac enhancer signals and strong corresponding changes in geneexpression. These genes constitute a small fraction of the total genesin each Hallmark gene set (5-10%), but highlight key genes within thesebiological processes where CDK19 can epigenetically regulate genetranscription.

4.7 Example 7 - Effects of CDK19 Knockdown on the Growth ofPre-Established Organoids

We explored the effect of CDK19 knockdown on the growth ofpre-established organoids in vitro and in pre-established PDX tumors invivo. This aimed to model the treatment of patients’ pre-existingtumors. In vitro, adding doxycycline to the treatment group (to induceCDK19 shRNA) significantly reduced the number of pre-establishedorganoids compared to the control (no doxycycline) (FIGS. 4A and 4B). InFIGS. 4A and 4B, number of organoid colonies at Day 0 (FIG. 4A) and Day16 (FIG. 4B) after initiating doxycycline treatment is shown, ****P <0.0001; ns is P > 0.05 (mean ± s.d., n = 6, experiment performed twice,P values determined by unpaired t-test). In vivo, feeding doxycycline tomice with pre-established inducCDK19KD-PDX-T1 or inducCDK19KD-PDX-T3(PDX-T3 cells transduced with a doxycycline-inducible CDK19 knockdownconstruct) tumors significantly impacted the growth of these tumors(FIGS. 4C and 4D). In FIGS. 4C and 4D, the growth of pre-establishedtumors in the doxycycline fed NSG mice and control NSG mice are shownfor inducCDK19KD-PDX-T1, ****P < 0.0001 ; ***P < 0.001 (mean ± s.d., n =5, experiment performed twice, P values determined by unpaired t-test)(FIG. 4C) and inducCDK19KD-PDX-T3, ****P < 0.0001; ***P < 0.001 (mean ±s.d., n = 5, experiment performed once, P values determined by unpairedt-test) (FIG. 4D). CDK19 shRNA induced tumors were ultimately 82%smaller in inducCDK19KD-PDX-T1 tumors and 38% smaller ininducCDK19KD-PDX-T3 tumors when compared to control tumors (FIGS. 4C and4D). In both inducCDK19KD-PDX-T1 and inducCDK19KD-PDX-T3 experiments,mouse total body weights were not significantly different between thetreatment and control groups (FIGS. 14A and 14B). Finally, survivalstudies showed that overall survival was significantly longer in micewhose PDX-T1 tumors were transduced with CDK19 shRNA compared to micetransduced with control shRNA (FIG. 4E). Shown in FIG. 4E areKaplan-Meir survival curves for mice engrafted with PDX-T1 xenograftstransduced with control shRNA (black line), shCDK19-1 (solid gray line)or shCDK19-2 (dashed gray line). Mice were followed with weeklymeasurements of tumor diameters. Mice were sacrificed when the longestdiameter of their tumor exceeded 17 mm. Two mice in the shCDK19-2 groupdid not develop PDX tumors and were sacrificed at the end of theexperiment. These mice were censored when constructing the survivalcurve for the shCDK19-2 group, ***P < 0.001 (n = 9, experiment performedthree times, log-rank (Mantel-Cox) test used to determine P values). Insummary, these experiments showed that even in pre-established tumors,specifically knocking down CDK19 can significantly decrease tumor growthand that CDK19 knockdown can prolong survival in mice.

4.8 Example 8 - Effects of CCT251921 on Pre-Established PDX Tumors

To model the use of a CDK19 targeted therapy clinically, we treated micewith pre-established PDX tumors with CCT251921 (FIG. 4F), an orallybioavailable inhibitor of both CDK19 and the closely related paralog,CDK8. PDX-T1 tumors were pre-established in mice before starting dailyoral administration (30 mg/kg) of CCT251921 or vehicle. Treatment withCCT251921 resulted in a significant reduction in tumor growth by day 14(FIG. 4G). Final volumes of the tumors in CCT251921 treated mice wereover 30% smaller than the tumors of vehicle treated mice (FIG. 4G). NSGmice with pre-established PDX-T1 xenograft tumors were treated withdaily oral gavage of CCT251921 or vehicle. Mice were followed with twiceweekly determinations of tumor volume, ****P < 0.0001; ***P < 0.001(mean ± s.d., n = 5, experiment performed once, P values determined byunpaired t-test). Mice in both the CCT251921 and vehicle cohortssuffered an overall weight loss, but this was not significantlydifferent between the two groups and most likely due to the effect ofdaily oral gavage on their feeding habits (FIG. 14C). It is well knownthat different biological outcomes can arise from gene knockdown versuschemical inhibition. We show here in pre-established tumors thatchemical inhibition of CDK19 kinase activity can recapitulate theeffects of total CDK19 loss shown in our knockdown studies.

From our data, we conclude that CDK19 regulates multiple cancer relevantpathways and that it is a potential therapeutic target in TICs. Thus,CDK19 inhibition is useful both to therapeutic strategies targetingtranscriptional co-factors such as CDK8, CDK9, and BRD4, and to thosetargeting TICs and their self-renewal pathways such as Hedgehog,Wnt/β-catenin, and Notch. However, some therapeutic approaches may belimited by toxicity caused to normal cells. This can be attributed tothe ubiquitous expression of transcriptional co-factors in normaltissues and the importance of self-renewal pathways in normal stemcells. BRD4 inhibition, for example, resulted in a disruption of tissuehomeostasis in multiple organs in mice. Similarly, due to the challengeof narrow therapeutic indices, Hedgehog, Notch, and Wnt pathwayinhibitors have had limited clinical success thus far. The biology ofCDK19 points towards potential advantages as a therapeutic target.Compared to other ubiquitous transcriptional co-factors such as itsparalog CDK8, CDK9, and BRD4, CDK19 has more limited tissue distribution(see, e.g., Tsutsui et al., Genes to cells : devoted to molecular &cellular mechanisms 16:1208-1218, 2011), potentially limiting thetoxicity from CDK19 inhibition, while CDK8, CDK9, and BRD4 knockouts arelethal (see, e.g., Brown et al., Mamm Genome 23:632-640, 2012;Westerling, Molecular and Cellular Biology 27:6177-6182, 2007; andHouzelstein et al., Molecular and Cellular Biology 22, 3794-3802, 2002).In addition, the limited expression of CDK19 in tissues could broadenthe therapeutic window to enable the otherwise toxic inhibition of stemcell pathways such as NOTCH, or critical processes, such as G2/Mcheckpoint. Our studies showingthat small molecule inhibition of CDK19impaired PDX growth affirms the potential of therapeutically targetingCDK19 in TNBC.

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37 Takebe, N. et al. Targeting Notch, Hedgehog, and Wnt pathways incancer stem cells: clinical update. Nat Rev Clin Oncol 12, 445-464,doi:10.1038/nrclinonc.2015.61 (2015).

38 Westerling, T., Kuuluvainen, E. & Makela, T. P. Cdk8 is essential forpreimplantation mouse development. Molecular and cellular biology 27,6177-6182, doi:10.1128/MCB.01302-06 (2007).

39 Houzelstein, D. et al. Growth and early postimplantation defects inmice deficient for the bromodomain-containing protein Brd4. Molecularand cellular biology 22, 3794-3802 (2002).

40 Schindelin, J. et al. Fiji: an open-source platform forbiological-image analysis. Nat Methods 9, 676-682,doi:10.1038/nmeth.2019 (2012).

41 Gao, J. et al. Integrative analysis of complex cancer genomics andclinical profiles using the cBioPortal. Sci Signal 6, pl1,doi:10.1126/scisignal.2004088 (2013).

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by thoseskilled in the relevant arts, once they have been made familiar withthis disclosure, that various changes in form and detail can be madewithout departing from the true scope of the invention in the appendedclaims. The invention is therefore not to be limited to the exactcomponents or details of methodology or construction set forth above.Except to the extent necessary or inherent in the processes themselves,no particular order to steps or stages of methods or processes describedin this disclosure, including the Figures, is intended or implied. Inmany cases the order of process steps may be varied without changing thepurpose, effect, or import of the methods described.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents (patents,published patent applications, and unpublished patent applications) isnot intended as an admission that any such document is pertinent priorart, nor does it constitute any admission as to the contents or date ofthe same.

CDK19 Transcript Variant 1 (NM_015076.4) (SEQ ID NO: 12)

1 tgtggccgcc gaggagtccc ttgctgaagg cggaccgcgg agcggcgggc ggcgggcggc  61 gcgcgcgcgc gcgcgagagg cggctgttgg agaagtggag cggcggtcgc ggggggagga 121 ggaggaggga ctgagcggcg gcggcccccg cgtcccgtgc ctctatgggg gaagcagaca 181 atggattatg atttcaaggc gaagctggcg gcggagcggg agcgggtgga ggatttgttt 241 gagtacgaag ggtgcaaagt gggacgcggc acctacggtc acgtctacaa ggcgaggcgg 301 aaagatggaa aagatgaaaa ggaatatgca ttgaagcaaa ttgaaggcac aggaatatcc 361 atgtcggctt gtagagagat tgcacttttg cgagaattga agcaccctaa tgtgattgca 421 ttgcagaagg tgttcctttc tcacagtgac aggaaggtat ggctgctgtt tgattatgca 481 gagcatgact tgtggcatat tattaagttt caccgtgcat caaaagcaaa taaaaagccc 541 atgcagttgc caagatctat ggttaaatcc ttactttacc agattcttga tggtatccat 601 tacctccatg caaattgggt gcttcacaga gacttgaaac cagcaaatat cctagtaatg 661 ggagaaggtc ctgagagggg gagagtcaaa atagctgaca tgggttttgc cagattattc 721 aattctcctc taaagccact agcagatttg gatccagtag ttgtgacatt ttggtatcgg 781 gctccagaac ttttgcttgg tgcaaggcat tatacaaagg ccattgatat atgggcaata 841 ggttgtatat ttgctgaatt gttgacttcg gaacctattt ttcactgtcg tcaggaagat 901 ataaaaacaa gcaatccctt tcatcatgat caactggatc ggatatttag tgtcatgggg 961 tttcctgcag ataaagactg ggaagatatt agaaagatgc cagaatatcc cacacttcaa1021 aaagacttta gaagaacaac gtatgccaac agtagcctca taaagtacat ggagaaacac1081 aaggtcaagc ctgacagcaa agtgttcctc ttgcttcaga aactcctgac catggatcca1141 accaagagaa ttacctcgga gcaagctctg caggatccct attttcagga ggaccctttg1201 ccaacattag atgtatttgc cggctgccag attccatacc ccaaacgaga attccttaat1261 gaagatgatc ctgaagaaaa aggtgacaag aatcagcaac agcagcagaa ccagcatcag1321 cagcccacag cccctccaca gcaggcagca gcccctccac aggcgccccc accacagcag1381 aacagcaccc agaccaacgg gaccgcaggt ggggctgggg ccggggtcgg gggcaccgga1441 gcagggttgc agcacagcca ggactccagc ctgaaccagg tgcctccaaa caagaagcca1501 cggctagggc cttcaggcgc aaactcaggt ggacctgtga tgccctcgga ttatcagcac1561 tccagttctc gcctgaatta ccaaagcagc gttcagggat cctctcagtc ccagagcaca1621 cttggctact cttcctcgtc tcagcagagc tcacagtacc acccatctca ccaggcccac1681 cggtactgac cagctcccgt tgggccaggc cagcccagcc cagagcacag gctccagcaa1741 tatgtctgca ttgaaaagaa ccaaaaaaat gcaaactatg atgccattta aaactcatac1801 acatgggagg aaaaccttat atactgagca ttgtgcagga ctgatagctc ttctttattg1861 acttaaagaa gattcttgtg aagtttcccc agcacccctt ccctgcatgt gttccattgt1921 gacttctctg ataaagcgtc tgatctaatc ccagcacttc tgtaaccttc agcatttctt1981 tgaaggattt cctggtgcac ctttctcatg ctgtagcaat cactatggtt tatcttttca2041 aagctctttt aataggattt taatgtttta gaaacaggat tccagtggtg tatagtttta2101 tacttcatga actgatttag caacacaggt aaaaatgcac cttttaaagc actacgtttt2161 cacagacaat aactgttctg ctcatggaag tcttaaacag aaactgttac tgtcccaaag2221 tactttacta ttacgttcgt atttatctag tttcagggaa ggtctaataa aaagacaagc2281 ggtgggacag agggaaccta caaccaaaaa ctgcctagat ctttgcagtt atgtgcttta2341 tgccacgaag aactgaagta tgtggtaatt tttatagaat cattcatatg gaactgagtt2401 cccagcatca tcttattctg aatagcattc agtaattaag aattacaatt ttaaccttca2461 tgtagctaag tctaccttaa aaagggtttc aagagctttg tacagtctcg atggcccaca2521 ccaaaacgct gaagagagta acaactgcac taggatttct gtaaggagta attttgatca2581 aaagacgtgt tacttccctt tgaaggaaaa gtttttagtg tgtattgtac ataaagtcgg2641 cttctctaaa gaaccattgg tttcttcaca tctgggtctg cgtgagtaac tttcttgcat2701 aatcaaggtt actcaagtag aagcctgaaa attaatctgc ttttaaaata aagagcagtg2761 ttctccattc gtatttgtat tagatataga gtgactattt ttaaagcatg ttaaaaattt2821 aggttttatt catgtttaaa gtatgtatta tgtatgcata attttgctgt tgttactgaa2881 acttaattct atcaagaatc tttttcattg cactgaatga tttcttttgc ccctaggaga2941 aaacttaata attgtgccta aaaactatgg gcggatagta taagactata ctagacaaag3001 tgaatatttg catttccatt atctatgaat tagtggctga gttctttctt agctgcttta3061 aggagcccct cactccccag agtcaaaagg aaatgtaaaa acttagagct cccattgtaa3121 tgtaaggggc aagaaatttg tgttcttctg aatgctacta gcagcaccag ccttgtttta3181 aatgttttct tgagctagaa gaaatagctg attattgtat atgcaaatta catgcatttt3241 taaaaactat tctttctgaa cttatctacc tggttatgat actgtgggtc catacacaag3301 taaaataaga ttagacagaa gccagtatac attttgcact attgatgtga tactgtagcc3361 agccaggacc ttactgatct cagcataata atgctcacta ataatgaagt ctgcatagtg3421 acactcatca agactgaaga tgaagcaggt tacgtgctcc attggaagga gtttctgata3481 gtctcctgct gttttacccc ttccattttt taaaataaga aattagcagc cctctgcata3541 atgtagctgc ctatatgcag ttttatcctg tgccctaaag cctcactgtc cagagctgtt3601 ggtcatcaga tgcttattgc accctcacca tgtgcctggt gccctgctgg gtagagaaca3661 cagaggacag ggcatacttc ttgtccttaa ggagcttgtg atctgtgaca gtaagccctc3721 ctgggatgtc tgtgccatgt gattgactta caagtgaaac tgtcttataa tatgaaggtc3781 tttttgttta cttctaaacc cacttgggta gttactatcc ccaaatctgt tctgtaaata3841 atattatgga agggtttcta tgtcagtcta ccttagagaa agccagtgat tcaatatcac3901 aaaaggcatt gacgtatctt tgaaatgttc acagcagcct tttaacaaca actgggtggt3961 ccttgtaggc agaacatact ctcctaagtg gttgtaggaa attgcaagga aaatagaagg4021 tctgttcttg ctctcaagga ggttaccttt aataaaagaa gacaaaccca gatagatatg4081 taaaccaaaa tactatgccc cttaatactt tataagcagc attgttaaat agttcttacg4141 cttatacatt cacagaacta ccctgttttc cttgtatata atgacttttg ctggcagaac4201 tgaaatataa actgtaaggg gatttcgtca gttgctccca gtatacaata tcctccagga4261 catagccaga aatctccatt ccacacatga ctgagttcct atccctgcac tggtactggc4321 tcttttctcc tctttccttg cctcagggtt cgtgctaccc actgattccc tttaccctta4381 gtaataattt tggatcattt tctttccttt aaaggggaac aaagcctttt ttttttttga4441 gacggagtgt tgctctgtca cccaagctgg agtgcagtgg cacgatcttg gctcactcca4501 acctccacct tccaggttca agtgattctc ctgcctcagc ctcccgagta gctgggacta4561 cgggcacgca ccaccacgtc tggctaattt ttgtattttt agtagagatg gggtttcacc4621 ctattggtca ggctggtctt gaattcctca cctcaggtca tccgcctgtc tcggcctccc4681 gaagtgctgg gattataggt gtgagccacc gcacccagtt gggaacaaag cctttttaac4741 acacgtaagg gccctcaaac cgtgggacct ctaaggagac ctttgaagct ttttgagggc4801 aaactttacc tttgtggtcc ccaaatgatg gcatttctct ttgaaattta ttagatactg4861 ttatgtcccc caagggtaca ggaggggcat ccctcagcct atgggaacac ccaaactagg4921 aggggttatt gacaggaagg aatgaatcca agtgaaggct ttctgctctt cgtgttacaa4981 accagtttca gagttagctt tctggggagg tgtgtgtttg tgaaaggaat tcaagtgttg5041 caggacagat gagctcaagg taaggtagct ttggcagcag ggctgatact atgaggctga5101 aacaatcctt gtgatgaagt agatcatgca gtgacataca aagaccaagg attatgtata5161 tttttatatc tctgtggttt tgaaacttta gtacttagaa ttttggcctt ctgcactact5221 cttttgctct tacgaacata atggactctt aagaatggaa agggatgaca tttacctatg5281 tgtgctgcct cattcctggt gaagcaactg ctacttgttc tctatgcctc taaaatgatg5341 ctgttttctc tgctaaaggt aaaagaaaag aaaaaaatag ttggaaaata agacatgcaa5401 cttgatgtgc ttttgagtaa atttatgcag cagaaactat acaatgaagg aagaattcta5461 tggaaattac aaatccaaaa ctctatgatg atgtcttcct agggagtaga gaaaggcagt5521 gaaatggcag ttagaccaac agaggcttga aggattcaag tacaagtaat attttgtata5581 aaacatagca gtttaggtcc ccataatcct caaaaatagt cacaaatata acaaagttca5641 ttgttttagg gtttttaaaa aacgtgttgt acctaaggcc atacttactc ttctatgcta5701 tcactgcaaa ggggtgatat gtatgtatta tataaaaaaa aaaaccctta atgcactgtt5761 atctcctaaa tatttagtaa attaatacta tttaattttt ttaaagattt gtctgtgtag5821 acactaaaag tattacacaa aatctggact gaaggtgtcc tttttaacaa caatttaaag5881 tactttttat atatgttatg tagtatatcc tttctaaact gcctagtttg tatattccta5941 taattcctat ttgtgaagtg tacctgttct tgtctctttt ttcagtcatt ttctgcacgc6001 atcccccttt atatggttat agagatgact gtagcttttc gtgctccact gcgaggtttg6061 tgctcagagc cgctgcaccc cagcgaggcc tgctccatgg agtgcaggac gagctactgc6121 tttggagcga gggtttcctg cttttgagtt gacctgactt ccttcttgaa atgactgtta6181 aaactaaaat aaattacatt gcatttattt tatattcttg gttgaaataa aatttaattg6241 actttg

CDK19 Transcript Variant 2 (NM_001300960.1) (SEQ ID NO: 13)

   1 tgtggccgcc gaggagtccc ttgctgaagg cggaccgcgg agcggcgggc ggcgggcggc  61 gcgcgcgcgc gcgcgagagg cggctgttgg agaagtggag cggcggtcgc ggggggagga 121 ggaggaggga ctgagcggcg gcggcccccg cgtcccgtgc ctctatgggg gaagcagaca 181 atggattatg atttcaaggc gaagctggcg gcggagcggg agcgggtgga ggatttgttt 241 gagtacgaag ggtgcaaagt gggacgcggc acctacggtc acgtctacaa ggcgaggcgg 301 aaagatggaa aagatgaaaa ggaatatgca ttgaagcaaa ttgaaggcac aggaatatcc 361 atgtcggctt gtagagagat tgcacttttg cgagaattga agcaccctaa tgtgattgca 421 ttgcagaagg tgttcctttc tcacagtgac aggaaggtat ggctgctgtt tgattatgca 481 gagcatgact tgtggcatat tattaagttt caccgtgcat caaaagcaaa taaaaagccc 541 atgcagttgc caagatctat ggttaaatcc ttactttacc agattcttga tggtatccat 601 tacctccatg caaattgggt gcttcacaga gacttgaaac cagcaaatat cctagtaatg 661 ggagaaggtc ctgagagggg gagagtcaaa atagatatat gggcaatagg ttgtatattt 721 gctgaattgt tgacttcgga acctattttt cactgtcgtc aggaagatat aaaaacaagc 781 aatccctttc atcatgatca actggatcgg atatttagtg tcatggggtt tcctgcagat 841 aaagactggg aagatattag aaagatgcca gaatatccca cacttcaaaa agactttaga 901 agaacaacgt atgccaacag tagcctcata aagtacatgg agaaacacaa ggtcaagcct 961 gacagcaaag tgttcctctt gcttcagaaa ctcctgacca tggatccaac caagagaatt1021 acctcggagc aagctctgca ggatccctat tttcaggagg accctttgcc aacattagat1081 gtatttgccg gctgccagat tccatacccc aaacgagaat tccttaatga agatgatcct1141 gaagaaaaag gtgacaagaa tcagcaacag cagcagaacc agcatcagca gcccacagcc1201 cctccacagc aggcagcagc ccctccacag gcgcccccac cacagcagaa cagcacccag1261 accaacggga ccgcaggtgg ggctggggcc ggggtcgggg gcaccggagc agggttgcag1321 cacagccagg actccagcct gaaccaggtg cctccaaaca agaagccacg gctagggcct1381 tcaggcgcaa actcaggtgg acctgtgatg ccctcggatt atcagcactc cagttctcgc1441 ctgaattacc aaagcagcgt tcagggatcc tctcagtccc agagcacact tggctactct1501 tcctcgtctc agcagagctc acagtaccac ccatctcacc aggcccaccg gtactgacca1561 gctcccgttg ggccaggcca gcccagccca gagcacaggc tccagcaata tgtctgcatt1621 gaaaagaacc aaaaaaatgc aaactatgat gccatttaaa actcatacac atgggaggaa1681 aaccttatat actgagcatt gtgcaggact gatagctctt ctttattgac ttaaagaaga1741 ttcttgtgaa gtttccccag caccccttcc ctgcatgtgt tccattgtga cttctctgat1801 aaagcgtctg atctaatccc agcacttctg taaccttcag catttctttg aaggatttcc1861 tggtgcacct ttctcatgct gtagcaatca ctatggttta tcttttcaaa gctcttttaa1921 taggatttta atgttttaga aacaggattc cagtggtgta tagttttata cttcatgaac1981 tgatttagca acacaggtaa aaatgcacct tttaaagcac tacgttttca cagacaataa2041 ctgttctgct catggaagtc ttaaacagaa actgttactg tcccaaagta ctttactatt2101 acgttcgtat ttatctagtt tcagggaagg tctaataaaa agacaagcgg tgggacagag2161 ggaacctaca accaaaaact gcctagatct ttgcagttat gtgctttatg ccacgaagaa2221 ctgaagtatg tggtaatttt tatagaatca ttcatatgga actgagttcc cagcatcatc2281 ttattctgaa tagcattcag taattaagaa ttacaatttt aaccttcatg tagctaagtc2341 taccttaaaa agggtttcaa gagctttgta cagtctcgat ggcccacacc aaaacgctga2401 agagagtaac aactgcacta ggatttctgt aaggagtaat tttgatcaaa agacgtgtta2461 cttccctttg aaggaaaagt ttttagtgtg tattgtacat aaagtcggct tctctaaaga2521 accattggtt tcttcacatc tgggtctgcg tgagtaactt tcttgcataa tcaaggttac2581 tcaagtagaa gcctgaaaat taatctgctt ttaaaataaa gagcagtgtt ctccattcgt2641 atttgtatta gatatagagt gactattttt aaagcatgtt aaaaatttag gttttattca2701 tgtttaaagt atgtattatg tatgcataat tttgctgttg ttactgaaac ttaattctat2761 caagaatctt tttcattgca ctgaatgatt tcttttgccc ctaggagaaa acttaataat2821 tgtgcctaaa aactatgggc ggatagtata agactatact agacaaagtg aatatttgca2881 tttccattat ctatgaatta gtggctgagt tctttcttag ctgctttaag gagcccctca2941 ctccccagag tcaaaaggaa atgtaaaaac ttagagctcc cattgtaatg taaggggcaa3001 gaaatttgtg ttcttctgaa tgctactagc agcaccagcc ttgttttaaa tgttttcttg3061 agctagaaga aatagctgat tattgtatat gcaaattaca tgcattttta aaaactattc3121 tttctgaact tatctacctg gttatgatac tgtgggtcca tacacaagta aaataagatt3181 agacagaagc cagtatacat tttgcactat tgatgtgata ctgtagccag ccaggacctt3241 actgatctca gcataataat gctcactaat aatgaagtct gcatagtgac actcatcaag3301 actgaagatg aagcaggtta cgtgctccat tggaaggagt ttctgatagt ctcctgctgt3361 tttacccctt ccatttttta aaataagaaa ttagcagccc tctgcataat gtagctgcct3421 atatgcagtt ttatcctgtg ccctaaagcc tcactgtcca gagctgttgg tcatcagatg3481 cttattgcac cctcaccatg tgcctggtgc cctgctgggt agagaacaca gaggacaggg3541 catacttctt gtccttaagg agcttgtgat ctgtgacagt aagccctcct gggatgtctg3601 tgccatgtga ttgacttaca agtgaaactg tcttataata tgaaggtctt tttgtttact3661 tctaaaccca cttgggtagt tactatcccc aaatctgttc tgtaaataat attatggaag3721 ggtttctatg tcagtctacc ttagagaaag ccagtgattc aatatcacaa aaggcattga3781 cgtatctttg aaatgttcac agcagccttt taacaacaac tgggtggtcc ttgtaggcag3841 aacatactct cctaagtggt tgtaggaaat tgcaaggaaa atagaaggtc tgttcttgct3901 ctcaaggagg ttacctttaa taaaagaaga caaacccaga tagatatgta aaccaaaata3961 ctatgcccct taatacttta taagcagcat tgttaaatag ttcttacgct tatacattca4021 cagaactacc ctgttttcct tgtatataat gacttttgct ggcagaactg aaatataaac4081 tgtaagggga tttcgtcagt tgctcccagt atacaatatc ctccaggaca tagccagaaa4141 tctccattcc acacatgact gagttcctat ccctgcactg gtactggctc ttttctcctc4201 tttccttgcc tcagggttcg tgctacccac tgattccctt tacccttagt aataattttg4261 gatcattttc tttcctttaa aggggaacaa agcctttttt ttttttgaga cggagtgttg4321 ctctgtcacc caagctggag tgcagtggca cgatcttggc tcactccaac ctccaccttc4381 caggttcaag tgattctcct gcctcagcct cccgagtagc tgggactacg ggcacgcacc4441 accacgtctg gctaattttt gtatttttag tagagatggg gtttcaccct attggtcagg4501 ctggtcttga attcctcacc tcaggtcatc cgcctgtctc ggcctcccga agtgctggga4561 ttataggtgt gagccaccgc acccagttgg gaacaaagcc tttttaacac acgtaagggc4621 cctcaaaccg tgggacctct aaggagacct ttgaagcttt ttgagggcaa actttacctt4681 tgtggtcccc aaatgatggc atttctcttt gaaatttatt agatactgtt atgtccccca4741 agggtacagg aggggcatcc ctcagcctat gggaacaccc aaactaggag gggttattga4801 caggaaggaa tgaatccaag tgaaggcttt ctgctcttcg tgttacaaac cagtttcaga4861 gttagctttc tggggaggtg tgtgtttgtg aaaggaattc aagtgttgca ggacagatga4921 gctcaaggta aggtagcttt ggcagcaggg ctgatactat gaggctgaaa caatccttgt4981 gatgaagtag atcatgcagt gacatacaaa gaccaaggat tatgtatatt tttatatctc5041 tgtggttttg aaactttagt acttagaatt ttggccttct gcactactct tttgctctta5101 cgaacataat ggactcttaa gaatggaaag ggatgacatt tacctatgtg tgctgcctca5161 ttcctggtga agcaactgct acttgttctc tatgcctcta aaatgatgct gttttctctg5221 ctaaaggtaa aagaaaagaa aaaaatagtt ggaaaataag acatgcaact tgatgtgctt5281 ttgagtaaat ttatgcagca gaaactatac aatgaaggaa gaattctatg gaaattacaa5341 atccaaaact ctatgatgat gtcttcctag ggagtagaga aaggcagtga aatggcagtt5401 agaccaacag aggcttgaag gattcaagta caagtaatat tttgtataaa acatagcagt5461 ttaggtcccc ataatcctca aaaatagtca caaatataac aaagttcatt gttttagggt5521 ttttaaaaaa cgtgttgtac ctaaggccat acttactctt ctatgctatc actgcaaagg5581 ggtgatatgt atgtattata taaaaaaaaa aacccttaat gcactgttat ctcctaaata5641 tttagtaaat taatactatt taattttttt aaagatttgt ctgtgtagac actaaaagta5701 ttacacaaaa tctggactga aggtgtcctt tttaacaaca atttaaagta ctttttatat5761 atgttatgta gtatatcctt tctaaactgc ctagtttgta tattcctata attcctattt5821 gtgaagtgta cctgttcttg tctctttttt cagtcatttt ctgcacgcat ccccctttat5881 atggttatag agatgactgt agcttttcgt gctccactgc gaggtttgtg ctcagagccg5941 ctgcacccca gcgaggcctg ctccatggag tgcaggacga gctactgctt tggagcgagg6001 gtttcctgct tttgagttga cctgacttcc ttcttgaaat gactgttaaa actaaaataa6061 attacattgc atttatttta tattcttggt tgaaataaaa tttaattgac tttg

CDK19 Transcript Variant 3 (NM_001300963.1) (SEQ ID NO: 14)

  1 gaggggcggc cctggtacgc aggcgcgcat gctttgtggg ggcgaggctg tggtggcccg  61 agattccagg agggcttcgt gtatggacct caagcgttgg aggtagcaga cttttcagca 121 gaagaaaaga tgaaaaggaa tatgcattga agcaaattga aggcacagga atatccatgt 181 cggcttgtag agagattgca cttttgcgag aattgaagca ccctaatgtg attgcattgc 241 agaaggtgtt cctttctcac agtgacagga aggtatggct gctgtttgat tatgcagagc 301 atgacttgtg gcatattatt aagtttcacc gtgcatcaaa agcaaataaa aagcccatgc 361 agttgccaag atctatggtt aaatccttac tttaccagat tcttgatggt atccattacc 421 tccatgcaaa ttgggtgctt cacagagact tgaaaccagc aaatatccta gtaatgggag 481 aaggtcctga gagggggaga gtcaaaatag ctgacatggg ttttgccaga ttattcaatt 541 ctcctctaaa gccactagca gatttggatc cagtagttgt gacattttgg tatcgggctc 601 cagaactttt gcttggtgca aggcattata caaaggccat tgatatatgg gcaataggtt 661 gtatatttgc tgaattgttg acttcggaac ctatttttca ctgtcgtcag gaagatataa 721 aaacaagcaa tccctttcat catgatcaac tggatcggat atttagtgtc atggggtttc 781 ctgcagataa agactgggaa gatattagaa agatgccaga atatcccaca cttcaaaaag 841 actttagaag aacaacgtat gccaacagta gcctcataaa gtacatggag aaacacaagg 901 tcaagcctga cagcaaagtg ttcctcttgc ttcagaaact cctgaccatg gatccaacca 961 agagaattac ctcggagcaa gctctgcagg atccctattt tcaggaggac cctttgccaa1021 cattagatgt atttgccggc tgccagattc cataccccaa acgagaattc cttaatgaag1081 atgatcctga agaaaaaggt gacaagaatc agcaacagca gcagaaccag catcagcagc1141 ccacagcccc tccacagcag gcagcagccc ctccacaggc gcccccacca cagcagaaca1201 gcacccagac caacgggacc gcaggtgggg ctggggccgg ggtcgggggc accggagcag1261 ggttgcagca cagccaggac tccagcctga accaggtgcc tccaaacaag aagccacggc1321 tagggccttc aggcgcaaac tcaggtggac ctgtgatgcc ctcggattat cagcactcca1381 gttctcgcct gaattaccaa agcagcgttc agggatcctc tcagtcccag agcacacttg1441 gctactcttc ctcgtctcag cagagctcac agtaccaccc atctcaccag gcccaccggt1501 actgaccagc tcccgttggg ccaggccagc ccagcccaga gcacaggctc cagcaatatg1561 tctgcattga aaagaaccaa aaaaatgcaa actatgatgc catttaaaac tcatacacat1621 gggaggaaaa ccttatatac tgagcattgt gcaggactga tagctcttct ttattgactt1681 aaagaagatt cttgtgaagt ttccccagca ccccttccct gcatgtgttc cattgtgact1741 tctctgataa agcgtctgat ctaatcccag cacttctgta accttcagca tttctttgaa1801 ggatttcctg gtgcaccttt ctcatgctgt agcaatcact atggtttatc ttttcaaagc1861 tcttttaata ggattttaat gttttagaaa caggattcca gtggtgtata gttttatact1921 tcatgaactg atttagcaac acaggtaaaa atgcaccttt taaagcacta cgttttcaca1981 gacaataact gttctgctca tggaagtctt aaacagaaac tgttactgtc ccaaagtact2041 ttactattac gttcgtattt atctagtttc agggaaggtc taataaaaag acaagcggtg2101 ggacagaggg aacctacaac caaaaactgc ctagatcttt gcagttatgt gctttatgcc2161 acgaagaact gaagtatgtg gtaattttta tagaatcatt catatggaac tgagttccca2221 gcatcatctt attctgaata gcattcagta attaagaatt acaattttaa ccttcatgta2281 gctaagtcta ccttaaaaag ggtttcaaga gctttgtaca gtctcgatgg cccacaccaa2341 aacgctgaag agagtaacaa ctgcactagg atttctgtaa ggagtaattt tgatcaaaag2401 acgtgttact tccctttgaa ggaaaagttt ttagtgtgta ttgtacataa agtcggcttc2461 tctaaagaac cattggtttc ttcacatctg ggtctgcgtg agtaactttc ttgcataatc2521 aaggttactc aagtagaagc ctgaaaatta atctgctttt aaaataaaga gcagtgttct2581 ccattcgtat ttgtattaga tatagagtga ctatttttaa agcatgttaa aaatttaggt2641 tttattcatg tttaaagtat gtattatgta tgcataattt tgctgttgtt actgaaactt2701 aattctatca agaatctttt tcattgcact gaatgatttc ttttgcccct aggagaaaac2761 ttaataattg tgcctaaaaa ctatgggcgg atagtataag actatactag acaaagtgaa2821 tatttgcatt tccattatct atgaattagt ggctgagttc tttcttagct gctttaagga2881 gcccctcact ccccagagtc aaaaggaaat gtaaaaactt agagctccca ttgtaatgta2941 aggggcaaga aatttgtgtt cttctgaatg ctactagcag caccagcctt gttttaaatg3001 ttttcttgag ctagaagaaa tagctgatta ttgtatatgc aaattacatg catttttaaa3061 aactattctt tctgaactta tctacctggt tatgatactg tgggtccata cacaagtaaa3121 ataagattag acagaagcca gtatacattt tgcactattg atgtgatact gtagccagcc3181 aggaccttac tgatctcagc ataataatgc tcactaataa tgaagtctgc atagtgacac3241 tcatcaagac tgaagatgaa gcaggttacg tgctccattg gaaggagttt ctgatagtct3301 cctgctgttt taccccttcc attttttaaa ataagaaatt agcagccctc tgcataatgt3361 agctgcctat atgcagtttt atcctgtgcc ctaaagcctc actgtccaga gctgttggtc3421 atcagatgct tattgcaccc tcaccatgtg cctggtgccc tgctgggtag agaacacaga3481 ggacagggca tacttcttgt ccttaaggag cttgtgatct gtgacagtaa gccctcctgg3541 gatgtctgtg ccatgtgatt gacttacaag tgaaactgtc ttataatatg aaggtctttt3601 tgtttacttc taaacccact tgggtagtta ctatccccaa atctgttctg taaataatat3661 tatggaaggg tttctatgtc agtctacctt agagaaagcc agtgattcaa tatcacaaaa3721 ggcattgacg tatctttgaa atgttcacag cagcctttta acaacaactg ggtggtcctt3781 gtaggcagaa catactctcc taagtggttg taggaaattg caaggaaaat agaaggtctg3841 ttcttgctct caaggaggtt acctttaata aaagaagaca aacccagata gatatgtaaa3901 ccaaaatact atgcccctta atactttata agcagcattg ttaaatagtt cttacgctta3961 tacattcaca gaactaccct gttttccttg tatataatga cttttgctgg cagaactgaa4021 atataaactg taaggggatt tcgtcagttg ctcccagtat acaatatcct ccaggacata4081 gccagaaatc tccattccac acatgactga gttcctatcc ctgcactggt actggctctt4141 ttctcctctt tccttgcctc agggttcgtg ctacccactg attcccttta cccttagtaa4201 taattttgga tcattttctt tcctttaaag gggaacaaag cctttttttt ttttgagacg4261 gagtgttgct ctgtcaccca agctggagtg cagtggcacg atcttggctc actccaacct4321 ccaccttcca ggttcaagtg attctcctgc ctcagcctcc cgagtagctg ggactacggg4381 cacgcaccac cacgtctggc taatttttgt atttttagta gagatggggt ttcaccctat4441 tggtcaggct ggtcttgaat tcctcacctc aggtcatccg cctgtctcgg cctcccgaag4501 tgctgggatt ataggtgtga gccaccgcac ccagttggga acaaagcctt tttaacacac4561 gtaagggccc tcaaaccgtg ggacctctaa ggagaccttt gaagcttttt gagggcaaac4621 tttacctttg tggtccccaa atgatggcat ttctctttga aatttattag atactgttat4681 gtcccccaag ggtacaggag gggcatccct cagcctatgg gaacacccaa actaggaggg4741 gttattgaca ggaaggaatg aatccaagtg aaggctttct gctcttcgtg ttacaaacca4801 gtttcagagt tagctttctg gggaggtgtg tgtttgtgaa aggaattcaa gtgttgcagg4861 acagatgagc tcaaggtaag gtagctttgg cagcagggct gatactatga ggctgaaaca4921 atccttgtga tgaagtagat catgcagtga catacaaaga ccaaggatta tgtatatttt4981 tatatctctg tggttttgaa actttagtac ttagaatttt ggccttctgc actactcttt5041 tgctcttacg aacataatgg actcttaaga atggaaaggg atgacattta cctatgtgtg5101 ctgcctcatt cctggtgaag caactgctac ttgttctcta tgcctctaaa atgatgctgt5161 tttctctgct aaaggtaaaa gaaaagaaaa aaatagttgg aaaataagac atgcaacttg5221 atgtgctttt gagtaaattt atgcagcaga aactatacaa tgaaggaaga attctatgga5281 aattacaaat ccaaaactct atgatgatgt cttcctaggg agtagagaaa ggcagtgaaa5341 tggcagttag accaacagag gcttgaagga ttcaagtaca agtaatattt tgtataaaac5401 atagcagttt aggtccccat aatcctcaaa aatagtcaca aatataacaa agttcattgt5461 tttagggttt ttaaaaaacg tgttgtacct aaggccatac ttactcttct atgctatcac5521 tgcaaagggg tgatatgtat gtattatata aaaaaaaaaa cccttaatgc actgttatct5581 cctaaatatt tagtaaatta atactattta atttttttaa agatttgtct gtgtagacac5641 taaaagtatt acacaaaatc tggactgaag gtgtcctttt taacaacaat ttaaagtact5701 ttttatatat gttatgtagt atatcctttc taaactgcct agtttgtata ttcctataat5761 tcctatttgt gaagtgtacc tgttcttgtc tcttttttca gtcattttct gcacgcatcc5821 ccctttatat ggttatagag atgactgtag cttttcgtgc tccactgcga ggtttgtgct5881 cagagccgct gcaccccagc gaggcctgct ccatggagtg caggacgagc tactgctttg5941 gagcgagggt ttcctgcttt tgagttgacc tgacttcctt cttgaaatga ctgttaaaac6001 taaaataaat tacattgcat ttattttata ttcttggttg aaataaaatt taattgactt6061 tg

CDK19 Transcript Variant 4 (NM_001300964.1) (SEQ ID NO: 15)

   1 agaaaagaaa caagctgcgg tacaactgtc ctcaccagcc ctcgcctccc gagtcactgc  61 agccaaccct tcagcaagaa aagatgaaaa ggaatatgca ttgaagcaaa ttgaaggcac 121 aggaatatcc atgtcggctt gtagagagat tgcacttttg cgagaattga agcaccctaa 181 tgtgattgca ttgcagaagg tgttcctttc tcacagtgac aggaaggtat ggctgctgtt 241 tgattatgca gagcatgact tgtggcatat tattaagttt caccgtgcat caaaagcaaa 301 taaaaagccc atgcagttgc caagatctat ggttaaatcc ttactttacc agattcttga 361 tggtatccat tacctccatg caaattgggt gcttcacaga gacttgaaac cagcaaatat 421 cctagtaatg ggagaaggtc ctgagagggg gagagtcaaa atagctgaca tgggttttgc 481 cagattattc aattctcctc taaagccact agcagatttg gatccagtag ttgtgacatt 541 ttggtatcgg gctccagaac ttttgcttgg tgcaaggcat tatacaaagg ccattgatat 601 atgggcaata ggttgtatat ttgctgaatt gttgacttcg gaacctattt ttcactgtcg 661 tcaggaagat ataaaaacaa gcaatccctt tcatcatgat caactggatc ggatatttag 721 tgtcatgggg tttcctgcag ataaagactg ggaagatatt agaaagatgc cagaatatcc 781 cacacttcaa aaagacttta gaagaacaac gtatgccaac agtagcctca taaagtacat 841 ggagaaacac aaggtcaagc ctgacagcaa agtgttcctc ttgcttcaga aactcctgac 901 catggatcca accaagagaa ttacctcgga gcaagctctg caggatccct attttcagga 961 ggaccctttg ccaacattag atgtatttgc cggctgccag attccatacc ccaaacgaga1021 attccttaat gaagatgatc ctgaagaaaa aggtgacaag aatcagcaac agcagcagaa1081 ccagcatcag cagcccacag cccctccaca gcaggcagca gcccctccac aggcgccccc1141 accacagcag aacagcaccc agaccaacgg gaccgcaggt ggggctgggg ccggggtcgg1201 gggcaccgga gcagggttgc agcacagcca ggactccagc ctgaaccagg tgcctccaaa1261 caagaagcca cggctagggc cttcaggcgc aaactcaggt ggacctgtga tgccctcgga1321 ttatcagcac tccagttctc gcctgaatta ccaaagcagc gttcagggat cctctcagtc1381 ccagagcaca cttggctact cttcctcgtc tcagcagagc tcacagtacc acccatctca1441 ccaggcccac cggtactgac cagctcccgt tgggccaggc cagcccagcc cagagcacag1501 gctccagcaa tatgtctgca ttgaaaagaa ccaaaaaaat gcaaactatg atgccattta1561 aaactcatac acatgggagg aaaaccttat atactgagca ttgtgcagga ctgatagctc1621 ttctttattg acttaaagaa gattcttgtg aagtttcccc agcacccctt ccctgcatgt1681 gttccattgt gacttctctg ataaagcgtc tgatctaatc ccagcacttc tgtaaccttc1741 agcatttctt tgaaggattt cctggtgcac ctttctcatg ctgtagcaat cactatggtt1801 tatcttttca aagctctttt aataggattt taatgtttta gaaacaggat tccagtggtg1861 tatagtttta tacttcatga actgatttag caacacaggt aaaaatgcac cttttaaagc1921 actacgtttt cacagacaat aactgttctg ctcatggaag tcttaaacag aaactgttac1981 tgtcccaaag tactttacta ttacgttcgt atttatctag tttcagggaa ggtctaataa2041 aaagacaagc ggtgggacag agggaaccta caaccaaaaa ctgcctagat ctttgcagtt2101 atgtgcttta tgccacgaag aactgaagta tgtggtaatt tttatagaat cattcatatg2161 gaactgagtt cccagcatca tcttattctg aatagcattc agtaattaag aattacaatt2221 ttaaccttca tgtagctaag tctaccttaa aaagggtttc aagagctttg tacagtctcg2281 atggcccaca ccaaaacgct gaagagagta acaactgcac taggatttct gtaaggagta2341 attttgatca aaagacgtgt tacttccctt tgaaggaaaa gtttttagtg tgtattgtac2401 ataaagtcgg cttctctaaa gaaccattgg tttcttcaca tctgggtctg cgtgagtaac2461 tttcttgcat aatcaaggtt actcaagtag aagcctgaaa attaatctgc ttttaaaata2521 aagagcagtg ttctccattc gtatttgtat tagatataga gtgactattt ttaaagcatg2581 ttaaaaattt aggttttatt catgtttaaa gtatgtatta tgtatgcata attttgctgt2641 tgttactgaa acttaattct atcaagaatc tttttcattg cactgaatga tttcttttgc2701 ccctaggaga aaacttaata attgtgccta aaaactatgg gcggatagta taagactata2761 ctagacaaag tgaatatttg catttccatt atctatgaat tagtggctga gttctttctt2821 agctgcttta aggagcccct cactccccag agtcaaaagg aaatgtaaaa acttagagct2881 cccattgtaa tgtaaggggc aagaaatttg tgttcttctg aatgctacta gcagcaccag2941 ccttgtttta aatgttttct tgagctagaa gaaatagctg attattgtat atgcaaatta3001 catgcatttt taaaaactat tctttctgaa cttatctacc tggttatgat actgtgggtc3061 catacacaag taaaataaga ttagacagaa gccagtatac attttgcact attgatgtga3121 tactgtagcc agccaggacc ttactgatct cagcataata atgctcacta ataatgaagt3181 ctgcatagtg acactcatca agactgaaga tgaagcaggt tacgtgctcc attggaagga3241 gtttctgata gtctcctgct gttttacccc ttccattttt taaaataaga aattagcagc3301 cctctgcata atgtagctgc ctatatgcag ttttatcctg tgccctaaag cctcactgtc3361 cagagctgtt ggtcatcaga tgcttattgc accctcacca tgtgcctggt gccctgctgg3421 gtagagaaca cagaggacag ggcatacttc ttgtccttaa ggagcttgtg atctgtgaca3481 gtaagccctc ctgggatgtc tgtgccatgt gattgactta caagtgaaac tgtcttataa3541 tatgaaggtc tttttgttta cttctaaacc cacttgggta gttactatcc ccaaatctgt3601 tctgtaaata atattatgga agggtttcta tgtcagtcta ccttagagaa agccagtgat3661 tcaatatcac aaaaggcatt gacgtatctt tgaaatgttc acagcagcct tttaacaaca3721 actgggtggt ccttgtaggc agaacatact ctcctaagtg gttgtaggaa attgcaagga3781 aaatagaagg tctgttcttg ctctcaagga ggttaccttt aataaaagaa gacaaaccca3841 gatagatatg taaaccaaaa tactatgccc cttaatactt tataagcagc attgttaaat3901 agttcttacg cttatacatt cacagaacta ccctgttttc cttgtatata atgacttttg3961 ctggcagaac tgaaatataa actgtaaggg gatttcgtca gttgctccca gtatacaata4021 tcctccagga catagccaga aatctccatt ccacacatga ctgagttcct atccctgcac4081 tggtactggc tcttttctcc tctttccttg cctcagggtt cgtgctaccc actgattccc4141 tttaccctta gtaataattt tggatcattt tctttccttt aaaggggaac aaagcctttt4201 ttttttttga gacggagtgt tgctctgtca cccaagctgg agtgcagtgg cacgatcttg4261 gctcactcca acctccacct tccaggttca agtgattctc ctgcctcagc ctcccgagta4321 gctgggacta cgggcacgca ccaccacgtc tggctaattt ttgtattttt agtagagatg4381 gggtttcacc ctattggtca ggctggtctt gaattcctca cctcaggtca tccgcctgtc4441 tcggcctccc gaagtgctgg gattataggt gtgagccacc gcacccagtt gggaacaaag4501 cctttttaac acacgtaagg gccctcaaac cgtgggacct ctaaggagac ctttgaagct4561 ttttgagggc aaactttacc tttgtggtcc ccaaatgatg gcatttctct ttgaaattta4621 ttagatactg ttatgtcccc caagggtaca ggaggggcat ccctcagcct atgggaacac4681 ccaaactagg aggggttatt gacaggaagg aatgaatcca agtgaaggct ttctgctctt4741 cgtgttacaa accagtttca gagttagctt tctggggagg tgtgtgtttg tgaaaggaat4801 tcaagtgttg caggacagat gagctcaagg taaggtagct ttggcagcag ggctgatact4861 atgaggctga aacaatcctt gtgatgaagt agatcatgca gtgacataca aagaccaagg4921 attatgtata tttttatatc tctgtggttt tgaaacttta gtacttagaa ttttggcctt4981 ctgcactact cttttgctct tacgaacata atggactctt aagaatggaa agggatgaca5041 tttacctatg tgtgctgcct cattcctggt gaagcaactg ctacttgttc tctatgcctc5101 taaaatgatg ctgttttctc tgctaaaggt aaaagaaaag aaaaaaatag ttggaaaata5161 agacatgcaa cttgatgtgc ttttgagtaa atttatgcag cagaaactat acaatgaagg5221 aagaattcta tggaaattac aaatccaaaa ctctatgatg atgtcttcct agggagtaga5281 gaaaggcagt gaaatggcag ttagaccaac agaggcttga aggattcaag tacaagtaat5341 attttgtata aaacatagca gtttaggtcc ccataatcct caaaaatagt cacaaatata5401 acaaagttca ttgttttagg gtttttaaaa aacgtgttgt acctaaggcc atacttactc5461 ttctatgcta tcactgcaaa ggggtgatat gtatgtatta tataaaaaaa aaaaccctta5521 atgcactgtt atctcctaaa tatttagtaa attaatacta tttaattttt ttaaagattt5581 gtctgtgtag acactaaaag tattacacaa aatctggact gaaggtgtcc tttttaacaa5641 caatttaaag tactttttat atatgttatg tagtatatcc tttctaaact gcctagtttg5701 tatattccta taattcctat ttgtgaagtg tacctgttct tgtctctttt ttcagtcatt5761 ttctgcacgc atcccccttt atatggttat agagatgact gtagcttttc gtgctccact5821 gcgaggtttg tgctcagagc cgctgcaccc cagcgaggcc tgctccatgg agtgcaggac5881 gagctactgc tttggagcga gggtttcctg cttttgagtt gacctgactt ccttcttgaa5941 atgactgtta aaactaaaat aaattacatt gcatttattt tatattcttg gttgaaataa6001 aatttaattg actttg

Cyclin dependent kinase 8 (CDK8), transcript variant 1 (NM_001260.2)(SEQ ID NO: 16)

   1 gagtgccctc cctcctcctc tctttgagga ggtaccggct gttgtgcggc tctgcccttc  61 tgtttgagtg tatgggagag tgagtgagtg agtgagtgtg agcgtgtgtg tgagagcgtg 121 aggcgtgagt gcgcgtgtga gaggacgaga gcccgcctgg ccgccccgcc gctcccgccg 181 cagcaggagc agaacgcgcg gccggagaga gcggcggagc cggcgcccag ggagcccgcg 241 gggacaaggg cagagacacc gctccccacc cccagccctc gtccctcggc tctccttcgc 301 cgggggatcc tccccgttcc tccacccccg gccggcctct gccccgccgt ccccctggat 361 gtccctggcg ctttcgcggg gcctcctcct gctcttgccg catcagtcgg gctggtgctg 421 cggccggcgg gcgtagagcg ggcgggttcc cgggggctgc ggctgcccgt gcttccccgg 481 tccccacccc tgccccccgg ccccccgacc cagctctccg gcctcagagg ctgtgacaat 541 ggactatgac tttaaagtga agctgagcag cgagcgggag cgggtcgagg acctgtttga 601 atacgagggc tgcaaagttg gccgaggcac ttatggtcac gtctacaaag ccaagaggaa 661 agatgggaag gatgataaag actatgcttt aaaacaaata gaaggaactg ggatctctat 721 gtcggcatgt agagaaatag cattacttcg agagcttaag catccaaacg tcatttctct 781 tcaaaaggtg tttctgtctc atgctgatag gaaggtgtgg cttctgtttg actatgctga 841 acatgacctc tggcatataa tcaagtttca cagagcttct aaagcaaaca agaagccagt 901 tcagttacct cggggaatgg tgaagtcact attatatcag atcctagatg gtattcacta 961 cctgcatgct aactgggtgt tgcacagaga tttgaaacct gctaatattt tagttatggg1021 tgaaggtcct gagcgaggaa gagtaaaaat tgctgacatg ggctttgccc gattatttaa1081 ttcacctttg aagcctttag cagatttgga tccagtggtt gttacattct ggtaccgagc1141 ccctgaacta cttcttggag caaggcatta taccaaagct attgatattt gggctatagg1201 gtgtatattt gcagaactac taacgtcaga accaatattt cactgtcgac aagaggacat1261 caaaactagt aatccttatc accatgacca gctggacaga atattcaatg taatgggatt1321 tcctgcagat aaagattggg aagatataaa aaagatgcct gaacattcaa cattaatgaa1381 agatttcaga agaaatacgt ataccaactg cagccttatc aagtatatgg aaaaacataa1441 agttaaacca gatagtaaag cattccactt gcttcagaag ctgcttacca tggacccaat1501 aaagcgaatt acctcagaac aggctatgca ggacccctat ttcttagaag acccacttcc1561 tacatcagac gtttttgccg gttgtcaaat cccttaccca aaacgagaat ttttaacgga1621 agaagaacct gatgacaaag gagacaaaaa gaaccagcag cagcagcagg gcaataacca1681 cactaatgga actggccacc cagggaatca agacagcagt cacacacagg gacccccgtt1741 gaagaaagtg agagttgttc ctcctaccac tacctcaggt ggacttatca tgacctcaga1801 ctatcagcgt tccaatccac atgctgccta tcccaaccct ggaccaagca catcacagcc1861 gcagagcagc atgggatact cagctacctc ccagcagcct ccacagtact cacatcagac1921 acatcggtac tgagctgcat cggaatcttg tccatgcact gttgcgaatg ctgcagggct1981 gactgtgcag ctctctgcgg gaacctggta tgggccatga gaatgtactg tacaaccaca2041 tcttcaaaat gtccagtagc caagttccac cacttttcac agattggggt agtggcttcc2101 aagttgtacc tattttggag ttagacttga aaagaaagtg ctagcacagt ttgtgttgtg2161 gatttgctac ttccatagtt tacttgacat ggttcagact gaccaatgca tttttttcag2221 tgacagtctg tagcagttga agctgtgaat gtgctagggg caagcatttg tctttgtatg2281 tggtgaattt tttcagtgta acaacattat ctgaccaata gtacacacac agacacaaag2341 tttaactggt acttgaaaca tacagtatat gttaacgaaa taaccaagac tcgaaatgag2401 attattttgg tacacctttc tttttagtgt cttatcagtg ggctgattca ttttctacat2461 taatcagtgt tttctgacca agaatattgc ttggattttt ttgaaagtac aaaaagccac2521 atagtttttc cagaaaggtt tcaaaactcc caaagattaa cttccaactt ataagtttgt2581 ttttattttc aatctatgac ttgactggta ttaaagctgc tatttgatag taattaaata2641 tgttgtcatt gatataaacc tgtttggttc agcaaacaaa ctaaaatgat tgtcatagac2701 agtgttttat ttttcctgtt ggtgttgctg atttgtgagc atgctttaag atgaaaaaag2761 catgaatgat aacttcctta aaaaggtgcg gcatccaatt caaatatttt cgtcctgatt2821 ttaaagctgg ttggtgtagt gctattaaaa tttcgttcag ttaattttcc ttttgaaaac2881 ttgttcgcac gttgtttagg gtgcccttac ttcagcaaag gagaaggagt aggagagcct2941 tagaattttt gaggaaaaaa aaacctataa catacaatgt actgtatcaa actattttac3001 atgaatgaca caagtattct gaataaaaaa taattgaaca ttgttaaaaa caaggtgtta3061 tgtaataaat ttatttttca taaatcaaaa aaaaaaaaaa a

Cyclin dependent kinase 8 (CDK8), transcript variant 2 (NM_001318368.1)(SEQ ID NO: 17)

   1 gagtgccctc cctcctcctc tctttgagga ggtaccggct gttgtgcggc tctgcccttc  61 tgtttgagtg tatgggagag tgagtgagtg agtgagtgtg agcgtgtgtg tgagagcgtg 121 aggcgtgagt gcgcgtgtga gaggacgaga gcccgcctgg ccgccccgcc gctcccgccg 181 cagcaggagc agaacgcgcg gccggagaga gcggcggagc cggcgcccag ggagcccgcg 241 gggacaaggg cagagacacc gctccccacc cccagccctc gtccctcggc tctccttcgc 301 cgggggatcc tccccgttcc tccacccccg gccggcctct gccccgccgt ccccctggat 361 gtccctggcg ctttcgcggg gcctcctcct gctcttgccg catcagtcgg gctggtgctg 421 cggccggcgg gcgtagagcg ggcgggttcc cgggggctgc ggctgcccgt gcttccccgg 481 tccccacccc tgccccccgg ccccccgacc cagctctccg gcctcagagg ctgtgacaat 541 ggactatgac tttaaagtga agctgagcag cgagcgggag cgggtcgagg acctgtttga 601 atacgagggc tgcaaagttg gccgaggcac ttatggtcac gtctacaaag ccaagaggaa 661 agatgggaag gatgataaag actatgcttt aaaacaaata gaaggaactg ggatctctat 721 gtcggcatgt agagaaatag cattacttcg agagcttaag catccaaacg tcatttctct 781 tcaaaaggtg tttctgtctc atgctgatag gaaggtgtgg cttctgtttg actatgctga 841 acatgacctc tggcatataa tcaagtttca cagagcttct aaagcaaaca agaagccagt 901 tcagttacct cggggaatgg tgaagtcact attatatcag atcctagatg gtattcacta 961 cctgcatgct aactgggtgt tgcacagaga tttgaaacct gctaatattt tagttatggg1021 tgaaggtcct gagcgaggaa gagtaaaaat tgctgacatg ggctttgccc gattatttaa1081 ttcacctttg aagcctttag cagatttgga tccagtggtt gttacattct ggtaccgagc1141 ccctgaacta cttcttggag caaggcatta taccaaagct attgatattt gggctatagg1201 gtgtatattt gcagaactac taacgtcaga accaatattt cactgtcgac aagaggacat1261 caaaactagt aatccttatc accatgacca gctggacaga atattcaatg taatgggatt1321 tcctgcagat aaagattggg aagatataaa aaagatgcct gaacattcaa cattaatgaa1381 agatttcaga agaaatacgt ataccaactg cagccttatc aagtatatgg aaaaacataa1441 agttaaacca gatagtaaag cattccactt gcttcagaag ctgcttacca tggacccaat1501 aaagcgaatt acctcagaac aggctatgca ggacccctat ttcttagaag acccacttcc1561 tacatcagac gtttttgccg gttgtcaaat cccttaccca aaacgagaat ttttaacgga1621 agaagaacct gatgacaaag gagacaaaaa ccagcagcag cagcagggca ataaccacac1681 taatggaact ggccacccag ggaatcaaga cagcagtcac acacagggac ccccgttgaa1741 gaaagtgaga gttgttcctc ctaccactac ctcaggtgga cttatcatga cctcagacta1801 tcagcgttcc aatccacatg ctgcctatcc caaccctgga ccaagcacat cacagccgca1861 gagcagcatg ggatactcag ctacctccca gcagcctcca cagtactcac atcagacaca1921 tcggtactga gctgcatcgg aatcttgtcc atgcactgtt gcgaatgctg cagggctgac1981 tgtgcagctc tctgcgggaa cctggtatgg gccatgagaa tgtactgtac aaccacatct2041 tcaaaatgtc cagtagccaa gttccaccac ttttcacaga ttggggtagt ggcttccaag2101 ttgtacctat tttggagtta gacttgaaaa gaaagtgcta gcacagtttg tgttgtggat2161 ttgctacttc catagtttac ttgacatggt tcagactgac caatgcattt ttttcagtga2221 cagtctgtag cagttgaagc tgtgaatgtg ctaggggcaa gcatttgtct ttgtatgtgg2281 tgaatttttt cagtgtaaca acattatctg accaatagta cacacacaga cacaaagttt2341 aactggtact tgaaacatac agtatatgtt aacgaaataa ccaagactcg aaatgagatt2401 attttggtac acctttcttt ttagtgtctt atcagtgggc tgattcattt tctacattaa2461 tcagtgtttt ctgaccaaga atattgcttg gatttttttg aaagtacaaa aagccacata2521 gtttttccag aaaggtttca aaactcccaa agattaactt ccaacttata agtttgtttt2581 tattttcaat ctatgacttg actggtatta aagctgctat ttgatagtaa ttaaatatgt2641 tgtcattgat ataaacctgt ttggttcagc aaacaaacta aaatgattgt catagacagt2701 gttttatttt tcctgttggt gttgctgatt tgtgagcatg ctttaagatg aaaaaagcat2761 gaatgataac ttccttaaaa aggtgcggca tccaattcaa atattttcgt cctgatttta2821 aagctggttg gtgtagtgct attaaaattt cgttcagtta attttccttt tgaaaacttg2881 ttcgcacgtt gtttagggtg cccttacttc agcaaaggag aaggagtagg agagccttag2941 aatttttgag gaaaaaaaaa cctataacat acaatgtact gtatcaaact attttacatg3001 aatgacacaa gtattctgaa taaaaaataa ttgaacattg ttaaaaacaa ggtgttatgt3061 aataaattta tttttcataa atcaaaaaaa aaaaaaaa

Cyclin dependent kinase 8 (CDK8), transcript variant 3 (NM_001346501.1)(SEQ ID NO: 18)

   1 gagtgccctc cctcctcctc tctttgagga ggtaccggct gttgtgcggc tctgcccttc  61 tgtttgagtg tatgggagag tgagtgagtg agtgagtgtg agcgtgtgtg tgagagcgtg 121 aggcgtgagt gcgcgtgtga gaggacgaga gcccgcctgg ccgccccgcc gctcccgccg 181 cagcaggagc agaacgcgcg gccggagaga gcggcggagc cggcgcccag ggagcccgcg 241 gggacaaggg cagagacacc gctccccacc cccagccctc gtccctcggc tctccttcgc 301 cgggggatcc tccccgttcc tccacccccg gccggcctct gccccgccgt ccccctggat 361 gtccctggcg ctttcgcggg gcctcctcct gctcttgccg catcagtcgg gctggtgctg 421 cggccggcgg gcgtagagcg ggcgggttcc cgggggctgc ggctgcccgt gcttccccgg 481 tccccacccc tgccccccgg ccccccgacc cagctctccg gcctcagagg ctgtgacaat 541 ggactatgac tttaaagtga agctgagcag cgagcgggag cgggtcgagg acctgtttga 601 atacgagggc tgcaaagttg gccgaggcac ttatggtcac gtctacaaag ccaagaggaa 661 agatgggaag gatgataaag actatgcttt aaaacaaata gaaggaactg ggatctctat 721 gtcggcatgt agagaaatag cattacttcg agagcttaag catccaaacg tcatttctct 781 tcaaaaggtg tttctgtctc atgctgatag gaaggtgtgg cttctgtttg actatgctga 841 acatgacctc tggcatataa tcaagtttca cagagcttct aaagcaaaca agaagccagt 901 tcagttacct cggggaatgg tgaagtcact attatatcag atcctagatg gtattcacta 961 cctgcatgct aactgggtgt tgcacagaga tttgctgaca tgggctttgc ccgattattt1021 aattcacctt tgaagccttt agcagatttg gatccagtgg ttgttacatt ctggtaccga1081 gcccctgaac tacttcttgg agcaaggcat tataccaaag ctattgatat ttgggctata1141 gggtgtatat ttgcagaact actaacgtca gaaccaatat ttcactgtcg acaagaggac1201 atcaaaacta gtaatcctta tcaccatgac cagctggaca gaatattcaa tgtaatggga1261 tttcctgcag ataaagattg ggaagatata aaaaagatgc ctgaacattc aacattaatg1321 aaagatttca gaagaaatac gtataccaac tgcagcctta tcaagtatat ggaaaaacat1381 aaagttaaac cagatagtaa agcattccac ttgcttcaga agctgcttac catggaccca1441 ataaagcgaa ttacctcaga acaggctatg caggacccct atttcttaga agacccactt1501 cctacatcag acgtttttgc cggttgtcaa atcccttacc caaaacgaga atttttaacg1561 gaagaagaac ctgatgacaa aggagacaaa aagaaccagc agcagcagca gggcaataac1621 cacactaatg gaactggcca cccagggaat caagacagca gtcacacaca gggacccccg1681 ttgaagaaag tgagagttgt tcctcctacc actacctcag gtggacttat catgacctca1741 gactatcagc gttccaatcc acatgctgcc tatcccaacc ctggaccaag cacatcacag1801 ccgcagagca gcatgggata ctcagctacc tcccagcagc ctccacagta ctcacatcag1861 acacatcggt actgagctgc atcggaatct tgtccatgca ctgttgcgaa tgctgcaggg1921 ctgactgtgc agctctctgc gggaacctgg tatgggccat gagaatgtac tgtacaacca1981 catcttcaaa atgtccagta gccaagttcc accacttttc acagattggg gtagtggctt2041 ccaagttgta cctattttgg agttagactt gaaaagaaag tgctagcaca gtttgtgttg2101 tggatttgct acttccatag tttacttgac atggttcaga ctgaccaatg catttttttc2161 agtgacagtc tgtagcagtt gaagctgtga atgtgctagg ggcaagcatt tgtctttgta2221 tgtggtgaat tttttcagtg taacaacatt atctgaccaa tagtacacac acagacacaa2281 agtttaactg gtacttgaaa catacagtat atgttaacga aataaccaag actcgaaatg2341 agattatttt ggtacacctt tctttttagt gtcttatcag tgggctgatt cattttctac2401 attaatcagt gttttctgac caagaatatt gcttggattt ttttgaaagt acaaaaagcc2461 acatagtttt tccagaaagg tttcaaaact cccaaagatt aacttccaac ttataagttt2521 gtttttattt tcaatctatg acttgactgg tattaaagct gctatttgat agtaattaaa2581 tatgttgtca ttgatataaa cctgtttggt tcagcaaaca aactaaaatg attgtcatag2641 acagtgtttt atttttcctg ttggtgttgc tgatttgtga gcatgcttta agatgaaaaa2701 agcatgaatg ataacttcct taaaaaggtg cggcatccaa ttcaaatatt ttcgtcctga2761 ttttaaagct ggttggtgta gtgctattaa aatttcgttc agttaatttt ccttttgaaa2821 acttgttcgc acgttgttta gggtgccctt acttcagcaa aggagaagga gtaggagagc2881 cttagaattt ttgaggaaaa aaaaacctat aacatacaat gtactgtatc aaactatttt2941 acatgaatga cacaagtatt ctgaataaaa aataattgaa cattgttaaa aacaaggtgt3001 tatgtaataa atttattttt cataaatcaa aaaaaaaaaa aaa

What is claimed is:
 1. A method of treating a patient diagnosed withtriple-negative breast cancer (TNBC), comprising administering atherapeutically effective dose of an agent that inhibits expression oractivity of cyclin-dependent kinase 19 (CDK19), wherein the agentcomprises a small molecule inhibitor of CDK19 activity, and whereinadministration of the agent results in at least one of a reduction incachexia, increase in survival time, elongation in time to tumorprogression, reduction in tumor mass, reduction in tumor burden,prolongation in time to tumor metastasis, a prolongation in time totumor recurrence, tumor response, complete response, partial response,stable disease, progressive disease, or progression free survival.
 2. Amethod of treating a patient diagnosed with triple-negative breastcancer (TNBC), wherein the cancer is characterized by a tumor comprisingEpCAM^(med/high) and CD10-^(/low) epithelial cells, the methodcomprising administering a therapeutically effective dose of an agentthat inhibits cyclin-dependent kinase 19 (CDK19) expression or activity,wherein the agent is a small molecule inhibitor of CDK19 activity,wherein the treatment reduces the number of EpCAM^(med/high) andCD10-^(/low) cells in the tumor, reduces to number of EpCAM^(med/high)and CD10-^(/low) cells per unit volume of the tumor, or results in areduction of the ratio of EpCAM^(med/high) and CD10-^(/low) epithelialcells to normal cells in the tumor.
 3. A method of reducing metastasisof TNBC in a patient, the method comprising administering atherapeutically effective dose of an agent that inhibits expression oractivity of CDK19, wherein the agent is a small molecule inhibitor ofCDK19 activity.
 4. The method of claim 1, wherein the patient is treatedwith a combination therapy comprising (a) an agent that inhibitsexpression or activity of CDK19 and (b) radiation therapy and/orchemotherapy.
 5. The method of claim 1, comprising detectingEpCAM^(med/high)/CD10-^(/low) cells in a tissue sample from the patientprior to or after initiating therapy.
 6. (canceled)
 7. The method of anyof claim 1 wherein the agent inhibits expression or activity of CDK19 toa greater extent than it inhibits expression or activity of CDK8.
 8. Themethod of claim 1 claim 1 wherein the agent is a small moleculeinhibitor that binds to the ATP binding site of CDK19 to inhibit itsactivity.
 9. The method of claim 1, wherein the agent binds to parts ofCDK19 outside of the ATP binding site.
 10. The method of claim 1,wherein the agent binds to CDK19 with a higher affinity than to CDK8.11. The method of claim 1 wherein the agent is a small moleculeinhibitor other than one or more compounds selected from the groupconsisting of Cortistatin A, Sorafenib, Linifanib, Ponatinib, Senexin B,CCT251545, and CCT251921. 12-15. (canceled)
 16. The method of claim 1,wherein the agent binds CDK 19 in the cytoplasm of a breast epithelialcell.
 17. A method of predicting the likely therapeutic responsivenessof a subject with TNBC to the method of treatment of claim 1 comprising:(a) quantitating EpCAM^(med/high)/CD10-^(/low) cells in a tumor sampleobtained from the subject; (b) comparing the quantity ofEpCAM^(med/high)/CD10′^(/low) cells in (a) to a reference valuecharacteristic of tumors responsive to a CDK19 targeting therapy, and(c) treating the patient with the agent that inhibits expression oractivity of cyclin-dependent kinase 19 (CDK19) if the quantity ofEpCAM^(med/high)/CD10-^(/low) cells is equal to or exceeds the referencevalue.
 18. The method of claim 2, wherein the agent inhibits expressionor activity of CDK 19 to a greater extent than it inhibits expression oractivity of CDK8.
 19. The method of claim 2, wherein the agent is asmall molecule inhibitor other than one or more compounds selected fromthe group consisting of Cortistatin A, Sorafenib, Linifanib, Ponatinib,Senexin B, CCT251545, and CCT251921.
 20. The method of claim 3,comprising detecting EpCAM^(med/high)/CD10′^(/low) cells in a tissuesample from the patient prior to the administering.
 21. The method ofclaim 3, wherein the agent inhibits expression or activity of CDK 19 toa greater extent than it inhibits expression or activity of CDK8. 22.The method of claim 3, wherein the agent binds to CDK19 with a higheraffinity than to CDK8.
 23. The method of claim 3, wherein the agent is asmall molecule inhibitor that binds to the ATP binding site of CDK19 toinhibit its activity.
 24. The method of claim 3, wherein the agent bindsto parts of CDK19 outside of the ATP binding site.
 25. The method ofclaim 3, wherein the agent is a small molecule inhibitor other than oneor more compounds selected from the group consisting of Cortistatin A,Sorafenib, Linifanib, Ponatinib, Senexin B, CCT251545, and CCT251921.